Immunomodulatory agent-polymeric compounds

ABSTRACT

This invention relates to compositions, and related compounds and methods, of conjugates of immunomodulatory agents and polymers or unit(s) thereof. The conjugates may be contained within synthetic nanocarriers, and the immunomodulatory agents may be released from the synthetic nanocarriers in a pH sensitive manner.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/138,601, filed Dec. 23, 2013, now allowed, which is a continuation ofU.S. patent application Ser. No. 12/788,266, filed May 26, 2010, nowU.S. Pat. No. 8,629,151, which claims the benefit under 35 U.S.C. §119of U.S. Provisional Application Nos. 61/217,129, 61/217,117, 61/217,124,and 61/217,116, each filed May 27, 2009, the contents of each of whichare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to compositions, and related compounds andmethods, of conjugates of immunomodulatory agents and polymers orunit(s) thereof. The conjugates may be contained within syntheticnanocarriers, and the immunomodulatory agents may be released from thesynthetic nanocarriers in a pH sensitive manner.

BACKGROUND

Immunomodulatory agents are used to produce immune responses insubjects. It is at times advantageous to attach such agents to deliveryvehicles. Currently, known attachment chemistries often require certainreactive groups, utilize certain activation steps for attachment tooccur, and/or result in conjugates that do not exhibit optimalproperties. There is a need, therefore, for new methods for theattachment of immunomodulatory agents to delivery vehicles as well as aneed for the resulting conjugates that exhibit desired properties.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a compound that comprisesa structure as in formula (I):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; and R₈ is abiodegradable polymer or unit thereof. In one embodiment, for thecompound of formula (I), the biodegradable polymer or unit thereofcomprises a polyester, polycarbonate, or a polyamide, or unit thereof.In another embodiment, the biodegradable polymer or unit thereofcomprises poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid), or polycaprolactone, or unit thereof.

In another aspect, the present invention provides a compound thatcomprises a structure as in formula (II):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; R₅ is a polymeror unit thereof; X=C, N, O, or S; R₆ and R₇ are each independentlyabsent, H, or substituted; and R₉, R₁₀, R₁₁, and R₁₂ are eachindependently H, a halogen, OH, thio, NH₂, or substituted orunsubstituted alkyl, aryl, heterocyclic, alkoxy, aryloxy, alkylthio,arylthio, alkylamino, or arylamino. In a further embodiment, for thecompound of formula (II), the polymer or unit thereof comprises apolyester, polycarbonate, polyamide, or a polyether, or unit thereof. Inanother embodiment, the polymer or unit thereof comprises poly(lacticacid), poly(glycolic acid), poly(lactic-co-glycolic acid),polycaprolactone, or poly(ethylene glycol), or unit thereof. In yetanother embodiment, the polymer is biodegradable.

In one embodiment, for a compound of formula (I) or (II), R₁ is H, R₂ isisobutyl, Y is C, and R₃ and R₄ are combined to form a benzene ring withthe carbon atoms of the pyridine ring to which they are connected. Inanother embodiment, R₁ is ethoxymethyl, R₂ is hydroxyisobutyl, Y=C, andR₃ and R₄ are combined to form a benzene ring with the carbon atoms ofthe pyridine ring to which they are connected. In yet anotherembodiment, R₁ is ethoxymethyl, R₂ is methanesulfonamidoisobutyl, Y=C,and R₃ and R₄ are combined to form a benzene ring with the carbon atomsof the pyridine ring to which they are connected. In one embodiment, R₁is OH, R₂ is benzyl, Y=N, R₃ is absent, and R₄ is butoxy. In anotherembodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ isbutylamino. In yet another embodiment, Y is N, R₁ is OH, R₂ is benzyl,R₃ is absent, and R₄ is butoxy. In still yet another embodiment, Y is N,R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ is benzylamino. In oneembodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ ispentyl.

In one embodiment, for a compound of formula (I) or (II), the polymer isinsoluble in water at pH=7.4 and at 25° C. In another embodiment, for acompound of formula (I) or (II), the polymer is insoluble in water atpH=7.4 and at 25° C. but soluble at pH=4.5 and at 25° C. In oneembodiment, for a compound of formula (I) or (II), the polymer has aweight average molecular weight ranging from 800 Daltons to 10,000Daltons, as determined using gel permeation chromatography. In anotherembodiment, for a compound of formula (I) or (II), the polymer or unitthereof does not comprise polyketal or unit thereof. In one embodiment,a composition is provided comprising a compound having a formula (I) or(II). In a further embodiment, the composition further comprises apharmaceutically acceptable excipient.

In one embodiment, a synthetic nanocarrier is provided that comprisesthe compound having a formula (I) or (II). In a further embodiment, thesynthetic nanocarrier further comprises a B cell antigen and/or a T cellantigen. In yet another embodiment, the synthetic nanocarrier furthercomprises an antigen presenting cell (APC) targeting feature. In afurther embodiment, the synthetic nanocarrier is a dendrimer, buckyball,nanowire, peptide or protein-based nanoparticle, nanoparticle thatcomprises a combination of nanomaterials, spheroidal nanoparticle, cubicnanoparticle, pyramidal nanoparticle, oblong nanoparticle, cylindricalnanoparticle, or toroidal nanoparticle. In another embodiment, acomposition is provided comprising a synthetic nanocarrier. In yet afurther embodiment, the composition further comprises a pharmaceuticallyacceptable excipient.

In one embodiment, a composition comprising a vaccine comprising acompound of formula (I) or (II) is provided. In another embodiment, acomposition comprising a vaccine comprising a composition comprising acompound of formula (I) or (II) is provided. In yet another embodiment,a composition comprising a vaccine comprising the synthetic nanocarriercomprising a compound of formula (I) or (II) is provided. In still yetanother embodiment, a method comprises a administering to a subject anyof the above described compounds, compositions, or synthetic nanocarrieris provided. In a further embodiment, an immune response is induced orenhanced in the subject following administering to a subject any of theabove described compounds, compositions, or synthetic nanocarrier.

In one aspect, a method for making a conjugate that comprises astructure as in formula (I):

comprises: activating a biodegradable polymer or unit thereof, andexposing the activated biodegradable polymer or unit thereof and acompound comprising a structure as in formula (III) to a base and/orsolvent:

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; and R₈ is abiodegradable polymer or unit thereof. In one embodiment, thebiodegradable polymer or unit thereof comprises a polyester,polycarbonate, or a polyamide, or unit thereof. In a further embodiment,the biodegradable polymer or unit thereof comprises poly(lactic acid),poly(glycolic acid), poly(lactic-co-glycolic acid), or polycaprolactone,or unit thereof.

In another aspect, a method for making a conjugate that comprises astructure as in formula (I):

comprises exposing a composition comprising a polymer or unit thereofand a compound comprising a structure as in formula (III) to a couplingagent and base and/or solvent:

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; and R₈ is apolymer or unit thereof.

In another aspect, a method for making a conjugate that comprises astructure as in formula (II):

comprises combining an alcohol, a catalyst, and a compound comprising astructure as in formula (IV):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; R₅ is a polymeror unit thereof; X is C, N, O, or S; R₆ and R₇ are each independently Hor substituted; and R₉, R₁₀, R₁₁, and R₁₂ are each independently H, ahalogen, OH, thio, NH₂, or substituted or unsubstituted alkyl, aryl,heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, orarylamino; and heating the alcohol, catalyst, and compound. In someembodiment, the alcohol, catalyst, and compound are heated in thepresence of a solvent.

In yet another aspect, a method for making a conjugate that comprises astructure as in formula (II):

comprises combining an alcohol and a compound comprising a structure asin formula (IV):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; R₅ is a polymeror unit thereof; X is C, N, O, or S; R₆ and R₇ are each independently Hor substituted; and R₉, R₁₀, R₁₁, and R₁₂ are each independently H, ahalogen, OH, thio, NH₂, or substituted or unsubstituted alkyl, aryl,heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, orarylamino; heating the alcohol and compound; and adding a catalyst. Inone embodiment, the alcohol, compound, and catalyst are heated whileand/or after the catalyst is added.

In yet another aspect, a method for making a conjugate that comprises astructure as in formula (II):

comprises combining an alcohol, a catalyst, and a compound comprising astructure as in formula (IV):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; R₅ is a polymeror unit thereof; X is C, N, O, or S; R₆ and R₇ are each independently Hor substituted; and R₉, R₁₀, R₁₁, and R₁₂ are each independently H, ahalogen, OH, thio, NH₂, or substituted or unsubstituted alkyl, aryl,heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, orarylamino.

In one embodiment, for a method of making a compound of formula (II),the alcohol is a polymer or unit thereof with a terminal hydroxyl group.In a further embodiment, the polymer or unit thereof comprises apolyester, polycarbonate, polyamide, or a polyether, or unit thereof. Inyet another embodiment, the polymer or unit thereof comprises,poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid),polycaprolactone, or poly(ethylene glycol), or unit thereof.

In one embodiment, for a method of making a compound of formula (II),the catalyst is a phosphazine base, 1,8-diazabicycloundec-7-ene,1,4,7-triazabicyclodecene, or N-methyl-1,4,7-triazabicyclodecene. Inanother embodiment, the polymer has a weight average molecular weightranging from 800 Daltons to 10,000 Daltons, as determined using gelpermeation chromatography. In yet another embodiment, the polymer isinsoluble in water at pH=7.4 and at 25° C. In another embodiment, thepolymer is insoluble in water at pH=7.4 and at 25° C. but soluble atpH=4.5 and at 25° C. In still yet another embodiment, the polymer orunit thereof does not comprise polyketal or unit thereof.

In one embodiment, for a method of making a compound of formula (II), R₁is H, R₂ is isobutyl, Y is C, and R₃ and R₄ are combined to form abenzene ring with the carbon atoms of the pyridine ring to which theyare connected. In another embodiment, R₁ is ethoxymethyl, R₂ ishydroxyisobutyl, Y=C, and R₃ and R₄ are combined to form a benzene ringwith the carbon atoms of the pyridine ring to which they are connected.In yet another embodiment, R₁ is ethoxymethyl, R₂ ismethanesulfonamidoisobutyl, Y=C, and R₃ and R₄ are combined to form abenzene ring with the carbon atoms of the pyridine ring to which theyare connected. In still yet another embodiment, R₁ is OH, R₂ is benzyl,Y=N, R₃ is absent, and R₄ is butoxy. In another embodiment, Y is N, R₁is OH, R₂ is benzyl, R₃ is absent, and R₄ is butylamino. In oneembodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ isbutoxy. In another embodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃ isabsent, and R₄ is benzylamino. In yet another embodiment, Y is N, R₁ isOH, R₂ is benzyl, R₃ is absent, and R₄ is pentyl.

In one aspect, the present invention provides a compound that comprisesa structure as in formula (IV):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; X is C, N, O, orS; R₆ and R₇ are each independently H or substituted; and R₉, R₁₀, R₁₁,and R₁₂ are each independently H, a halogen, OH, thio, NH₂, orsubstituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy, aryloxy,alkylthio, arylthio, alkylamino, or arylamino.

In one embodiment, for a compound of formula (IV), R₁ is H, R₂ isisobutyl, Y is C, and R₃ and R₄ are combined to form a benzene ring withthe carbon atoms of the pyridine ring to which they are connected. Inanother embodiment, R₁ is ethoxymethyl, R₂ is hydroxyisobutyl, Y=C, andR₃ and R₄ are combined to form a benzene ring with the carbon atoms ofthe pyridine ring to which they are connected. In yet anotherembodiment, R₁ is ethoxymethyl, R₂ is methanesulfonamidoisobutyl, Y=C,and R₃ and R₄ are combined to form a benzene ring with the carbon atomsof the pyridine ring to which they are connected. In still yet anotherembodiment, R₁ is OH, R₂ is benzyl, Y=N, R₃ is absent, and R₄ is butoxy.In one embodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄is butylamino. In another embodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃is absent, and R₄ is butoxy. In yet another embodiment, Y is N, R₁ isOH, R₂ is benzyl, R₃ is absent, and R₄ is benzylamino. In still yetanother embodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄is pentyl. In one embodiment, a composition is provided having acompound of formula (IV).

In one aspect, the present invention provides a method for making acompound that comprises a structure as in formula (IV):

comprising combining, in the presence of a solvent and/or heat, acompound that comprises a structure as in formula (III):

and a compound comprising a structure as in formula (V):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; X is C, N, O, orS; R₆ and R₇ are each independently H or substituted; and R₉, R₁₀, R₁₁,and R₁₂ are each independently H, a halogen, OH, thio, NH₂, orsubstituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy, aryloxy,alkylthio, arylthio, alkylamino, or arylamino.

In one embodiment, for a method comprising a compound of formula (IV),R₁ is H, R₂ is isobutyl, Y is C, and R₃ and R₄ are combined to form abenzene ring with the carbon atoms of the pyridine ring to which theyare connected. In another embodiment, R₁ is ethoxymethyl, R₂ ishydroxyisobutyl, Y=C, and R₃ and R₄ are combined to form a benzene ringwith the carbon atoms of the pyridine ring to which they are connected.In yet another embodiment, R₁ is ethoxymethyl, R₂ ismethanesulfonamidoisobutyl, Y=C, and R₃ and R₄ are combined to form abenzene ring with the carbon atoms of the pyridine ring to which theyare connected. In still yet another embodiment, R₁ is OH, R₂ is benzyl,Y=N, R₃ is absent, and R₄ is butoxy. In one embodiment, Y is N, R₁ isOH, R₂ is benzyl, R₃ is absent, and R₄ is butylamino. In anotherembodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ isbutoxy. In yet another embodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃ isabsent, and R₄ is benzylamino. In still yet another embodiment, Y is N,R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ is pentyl.

In one aspect, the present invention provides a method for making aconjugate that comprises a structure as in formula (VI):

comprising combining a catalyst, a diol having the formula (VII):HO-polymer-OH  (VII),and a compound comprising a structure as in formula (IV):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; X is C, N, O, orS; R₆ and R₇ are each independently H or substituted; and R₉, R₁₀, R₁₁,and R₁₂ are each independently H, a halogen, OH, thio, NH₂, orsubstituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy, aryloxy,alkylthio, arylthio, alkylamino, or arylamino; and heating the alcohol,catalyst, and compound. In one embodiment, for a method comprising acompound of formula (VI), the alcohol, catalyst, and compound are heatedin the presence of a solvent. In one embodiment of this aspect, thepolymer is intended to include a unit of a polymer provided herein.

In another aspect, the present invention provides a method for making aconjugate that comprises a structure as in formula (VI):

comprising combining a diol having the formula (VII):HO-polymer-OH  (VII),and a compound comprising a structure as in formula (IV):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; X is C, N, O, orS; R₆ and R₇ are each independently H or substituted; and R₉, R₁₀, R₁₁,and R₁₂ are each independently H, a halogen, OH, thio, NH₂, orsubstituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy, aryloxy,alkylthio, arylthio, alkylamino, or arylamino; heating the alcohol andcompound; and adding a catalyst. In a further embodiment, the alcohol,compound, and catalyst are heated while and/or after the catalyst isadded. In one embodiment of this aspect, the polymer is intended toinclude comprising a unit of a polymer provided herein.

In one aspect, the present invention provides a method for making aconjugate that comprises a structure as in formula (VI):

comprising combining, a catalyst, a diol having the formula (VII):HO-polymer-OH  (VII),and a compound comprising a structure as in formula (IV):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; X is C, N, O, orS; R₆ and R₇ are each independently H or substituted; and R₉, R₁₀, R₁₁,and R₁₂ are each independently H, a halogen, OH, thio, NH₂, orsubstituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy, aryloxy,alkylthio, arylthio, alkylamino, or arylamino.

In one embodiment, for a method of making a compound comprising theformula (VI), the compound of formula (VII) is selected from the groupconsisting polyketaldiols, poly(ethylene)glycol, polycaprolactone diol,diblock polylactide-co-poly(ethylene)glycol, diblockpolylactide/polyglycolide-co-poly(ethylene)glycol, diblockpolyglycolide-co-poly(ethylene)glycol, poly(propylene) glycol, andpoly(hexamethylene carbonate)diol. In one embodiment, for a method ofmaking a compound comprising the formula (VI), the catalyst is aphosphazine base, 1,8-diazabicycloundec-7-ene,1,4,7-triazabicyclodecene, or N-methyl-1,4,7-triazabicyclodecene. In afurther embodiment, the polymer has a weight average molecular weightranging from 800 Daltons to 10,000 Daltons, as determined using gelpermeation chromatography. In another embodiment, the polymer isinsoluble in water at pH=7.4 and at 25° C. In another embodiment, thepolymer is insoluble in water at pH=7.4 and at 25° C. but soluble atpH=4.5 and at 25° C. In yet another embodiment, the polymer does notcomprise polyketal or unit thereof.

In one embodiment, for a method of making a compound having the formula(VI), R₁ is H, R₂ is isobutyl, Y is C, and R₃ and R₄ are combined toform a benzene ring with the carbon atoms of the pyridine ring to whichthey are connected. In another embodiment, R₁ is ethoxymethyl, R₂ ishydroxyisobutyl, Y=C, and R₃ and R₄ are combined to form a benzene ringwith the carbon atoms of the pyridine ring to which they are connected.In yet another embodiment, R₁ is ethoxymethyl, R₂ ismethanesulfonamidoisobutyl, Y=C, and R₃ and R₄ are combined to form abenzene ring with the carbon atoms of the pyridine ring to which theyare connected. In still yet another embodiment, R₁ is OH, R₂ is benzyl,Y=N, R₃ is absent, and R₄ is butoxy. In one embodiment, Y is N, R₁ isOH, R₂ is benzyl, R₃ is absent, and R₄ is butylamino. In anotherembodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ isbutoxy. In yet another embodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃ isabsent, and R₄ is benzylamino. In still yet another embodiment, Y is N,R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ is pentyl.

In one aspect, a compound that comprises a structure as in formula (VI):

wherein each R₁, independently, =H, OH, SH, NH₂, or substituted orunsubstituted alkyl, alkoxy, alkylthio, or alkylamino;

each R₂, independently, =H, alkyl, or substituted alkyl;

each Y, independently, =N or C;

each R₃, independently, is absent if Y=N; or is H, alkyl, substitutedalkyl, or combined with R₄ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected if Y=C;

each R₄, independently, is H, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino when not combined with R₃ to form acarbocycle or heterocycle with the carbon atoms of the pyridine ring towhich they are connected; or is combined with R₃ to form a carbocycle orheterocycle with the carbon atoms of the pyridine ring to which they areconnected;

each X, independently, is C, N, O, or S;

each R₆ and R₇, independently, are each independently H or substituted;and

each R₉, R₁₀, R₁₁, and R₁₂, independently, are each independently H, ahalogen, OH, thio, NH₂, or substituted or unsubstituted alkyl, aryl,heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, orarylamino, is provided.

In one embodiment, the polymer is selected from the group consisting ofpolyketaldiols, poly(ethylene)glycol, polycaprolactone diol, diblockpolylactide-co-poly(ethylene)glycol, diblockpolylactide/polyglycolide-co-poly(ethylene)glycol, diblockpolyglycolide-co-poly(ethylene)glycol, poly(propylene) glycol,poly(hexamethylene carbonate)diol, and poly(tetrahydrofuran). In anotherembodiment of this aspect, the polymer includes a unit of a polymer. Inanother embodiment, the polymer has a weight average molecular weightranging from 800 Daltons to 10,000 Daltons, as determined using gelpermeation chromatography. In a further embodiment, the polymer isinsoluble in water at pH=7.4 and at 25° C. In yet another embodiment,the polymer does not comprise polyketal or unit thereof.

In one embodiment, R₁ is H, R₂ is isobutyl, Y is C, and R₃ and R₄ arecombined to form a benzene ring with the carbon atoms of the pyridinering to which they are connected. In another embodiment, R₁ isethoxymethyl, R₂ is hydroxyisobutyl, Y=C, and R₃ and R₄ are combined toform a benzene ring with the carbon atoms of the pyridine ring to whichthey are connected. In yet another embodiment, R₁ is ethoxymethyl, R₂ ismethanesulfonamidoisobutyl, Y=C, and R₃ and R₄ are combined to form abenzene ring with the carbon atoms of the pyridine ring to which theyare connected. In still another embodiment, R₁ is OH, R₂ is benzyl, Y=N,R₃ is absent, and R₄ is butoxy. In a further embodiment, Y is N, R₁ isOH, R₂ is benzyl, R₃ is absent, and R₄ is butylamino. In still anotherembodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ isbutoxy. In a further embodiment, Y is N, R₁ is OH, R₂ is benzyl, R₃ isabsent, and R₄ is benzylamino. In yet a further embodiment, Y is N, R₁is OH, R₂ is benzyl, R₃ is absent, and R₄ is pentyl.

In one embodiment, a composition comprising the above compounds isprovided. In another embodiment, the composition further comprises apharmaceutically acceptable excipient.

In another embodiment, a synthetic nanocarrier that comprises any of theforegoing compounds is provided. In one embodiment, the syntheticnanocarrier further comprises a B cell antigen and/or a T cell antigen.In another embodiment, the synthetic nanocarrier further comprises anantigen presenting cell (APC) targeting feature. In still anotherembodiment, the synthetic nanocarrier is a dendrimer, buckyball,nanowire, peptide or protein-based nanoparticle, nanoparticle thatcomprises a combination of nanomaterials, spheroidal nanoparticle, cubicnanoparticle, pyramidal nanoparticle, oblong nanoparticle, cylindricalnanoparticle, or toroidal nanoparticle.

In one embodiment, a composition comprising any of the foregoingsynthetic nanocarriers is provided. In one embodiment, the compositionfurther comprises a pharmaceutically acceptable excipient.

In another embodiment, a composition comprising a vaccine comprising anyof the foregoing compounds is provided. In yet another embodiment, acomposition comprising a vaccine comprising any of the foregoingcompositions is provided. In another embodiment, a compositioncomprising a vaccine comprising any of the foregoing syntheticnanocarriers is provided.

In another embodiment, a method comprising administering any of theforegoing compounds, compositions or synthetic nanocarriers to a subjectis provided. In one embodiment, the method is one where an immuneresponse is induced or enhanced in the subject.

In another aspect, a compound having a structure of any of the compoundsprovided herein is provided. Compositions, synthetic nanocarriers, andvaccines comprising any of the compounds provided are also provided.

In a further aspect, any of the methods of making a compound providedherein are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the release of resiquimod (R848) from synthetic nanocarrierformulations at pH 7.4, 37° C.

FIG. 2 shows the release of R848 from synthetic nanocarrier formulationsat pH 4.5, 37° C.

FIG. 3 shows the release of R848 from synthetic nanocarrier formulationsat pH 7.4 and pH 4.5 at 24 hours.

FIG. 4 shows the level of antibody induction by synthetic nanocarrierswith a CpG-containing immunostimulatory nucleic acid (Groups 2 and 3) ascompared to the level of antibody induction by synthetic nanocarrierswithout the CpG-containing immunostimulatory nucleic acid (Group 1).

FIG. 5 shows the level of antibody induction by synthetic nanocarriersthat release a phosphodiester, non-thioated CpG-containingimmunostimulatory nucleic acid or a thioated CpG-containingimmunostimulatory nucleic acid.

FIG. 6 shows the level of antibody induction by synthetic nanocarriersthat release R848 at different rates.

DETAILED DESCRIPTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified materials or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting of the use of alternativeterminology to describe the present invention.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyfor all purposes.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a polymer”includes a mixture of two or more such molecules, reference to “asolvent” includes a mixture of two or more such solvents, reference to“an adhesive” includes mixtures of two or more such materials, and thelike.

INTRODUCTION

The inventors have unexpectedly and surprisingly discovered that theproblems and limitations noted above can be overcome by practicing theinvention disclosed herein. In particular, the inventors haveunexpectedly discovered that it is possible to provide compounds,together with related compositions and methods, that comprise:

a structure as in formula (I):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino;

R₂=H, alkyl, or substituted alkyl;

Y=N or C;

R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, or combined withR₄ to form a carbocycle or heterocycle with the carbon atoms of thepyridine ring to which they are connected if Y=C;

R₄ is H, or substituted or unsubstituted alkyl, alkoxy, alkylthio, oralkylamino when not combined with R₃ to form a carbocycle or heterocyclewith the carbon atoms of the pyridine ring to which they are connected;or is combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; and

R₈ is a biodegradable polymer or unit thereof.

When using synthetic nanocarriers to produce an immune response in asubject, it is advantageous to include with the synthetic nanocarriersan immunomodulatory agent. Such an agent includes agents that areimmunomodulatory when uncoupled from the synthetic nanocarrier but maynot exhibit immunomodulatory properties when coupled to the syntheticnanocarrier. It is particularly advantageous to include theimmunomodulatory agent as part of the synthetic nanocarriers itself. Toachieve this, the immunomodulatory agent may be covalently attached toan appropriate polymer or unit thereof. It follows that the compoundsand conjugates provided herein, in some embodiments, comprise animmunomodulatory agent, which also is intended to include an agent thatis immunomodulatory when uncoupled from the polymer or unit thereof butthat may not exhibit immunomodulatory properties when coupled to thepolymer or unit thereof. The compounds provided herein can beincorporated into one or more synthetic nanocarriers. The compounds areincorporated into synthetic nanocarriers by methods known in the art ordescribed elsewhere herein.

In some embodiments, the polymer or unit thereof of the compounds orconjugates provided is a biodegradable polymer or unit thereof. Thepolymer or unit thereof, therefore, may comprise a polyester,polycarbonate, or polyamide, or unit thereof. It follows that thepolymer or unit thereof may comprise poly(lactic acid), poly(glycolicacid), poly(lactic-co-glycolic acid), or polycaprolactone, or unitthereof. Generally, it is preferred that if the polymer comprises apolyether, such as poly(ethylene glycol) (PEG) or unit thereof, thepolymer is a block-co-polymer of a polyether and a biodegradable polymersuch that the polymer is biodegradable. In some embodiments, the polymeror unit thereof does not comprise a polyether, such as poly(ethyleneglycol), or unit thereof. In other embodiments, the polymer does notsolely comprise a polyether or unit thereof, such as poly(ethyleneglycol), or unit thereof. Generally, for use as part of a syntheticnanocarrier the polymer of the compounds or conjugates provided hereinis insoluble in water at pH=7.4 and at 25° C., is biodegradable, orboth. In other embodiments, the polymer is insoluble in water at pH=7.4and at 25° C. but soluble at pH=4.5 and at 25° C. In still otherembodiments, the polymer is insoluble in water at pH=7.4 and at 25° C.but soluble at pH=4.5 and at 25° C. and biodegradable. The compounds,conjugates, and synthetic nanocarriers provided herein are unique incomposition and are useful for the preparation of vaccines andassociated materials.

Methods for making the aforementioned compounds are also provided. Inembodiments, a method for making a conjugate that comprises a structureas in formula (I):

comprises:

activating a biodegradable polymer or unit thereof, and

exposing the activated biodegradable polymer or unit thereof and acompound comprising a structure as in formula (III) to a base and/orsolvent:

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino;

R₂=H, alkyl, or substituted alkyl;

Y=N or C;

R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, or combined withR₄ to form a carbocycle or heterocycle with the carbon atoms of thepyridine ring to which they are connected if Y=C;

R₄ is H, or substituted or unsubstituted alkyl, alkoxy, alkylthio, oralkylamino when not combined with R₃ to form a carbocycle or heterocyclewith the carbon atoms of the pyridine ring to which they are connected;or is combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; and

R₈ is a biodegradable polymer or unit thereof.

In other embodiments, a method for making a conjugate that comprises astructure as in formula (I):

comprises:

exposing a composition comprising a polymer or unit thereof and acompound comprising a structure as in formula (III) to a coupling agentand base and/or solvent:

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino;

R₂=H, alkyl, or substituted alkyl;

Y=N or C;

R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, or combined withR₄ to form a carbocycle or heterocycle with the carbon atoms of thepyridine ring to which they are connected if Y=C;

R₄ is H, or substituted or unsubstituted alkyl, alkoxy, alkylthio, oralkylamino when not combined with R₃ to form a carbocycle or heterocyclewith the carbon atoms of the pyridine ring to which they are connected;or is combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; and

R₈ is a polymer or unit thereof.

The inventors have also unexpectedly discovered that it is possible toprovide compounds, together with related compositions and methods, thatcomprise:

a structure as in formula (II):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino;

R₂=H, alkyl, or substituted alkyl;

Y=N or C;

R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, or combined withR₄ to form a carbocycle or heterocycle with the carbon atoms of thepyridine ring to which they are connected if Y=C;

R₄ is H, or substituted or unsubstituted alkyl, alkoxy, alkylthio, oralkylamino when not combined with R₃ to form a carbocycle or heterocyclewith the carbon atoms of the pyridine ring to which they are connected;or is combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected;

R₅ is a polymer or unit thereof;

X=C, N, O, or S;

R₆ and R₇ are each independently absent, H, or substituted; and

R₉, R₁₀, R₁₁, and R₁₂ are each independently H, a halogen, OH, thio,NH₂, or substituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy,aryloxy, alkylthio, arylthio, alkylamino, or arylamino.

It has been discovered that it is possible to attach agents, such asimmunomodulatory agents, comprising a structure as in formula (III), toa polymer or unit thereof with a terminal alcohol. Generally, terminalalcohols are less reactive, making attachment chemistry problematic. Ithas been found that imides, such as those comprising a structure as informula (IV), will react with a terminal alcohol using catalystscommonly used in ring opening polymerizations. The resulting reactionproduct links the imide to the alcohol via an ester bond.

Accordingly, methods for making conjugates via the aforementionedchemistry are also provided. In some embodiments, a method for making aconjugate that comprises a structure as in formula (II):

comprises:

combining an alcohol, a catalyst, and a compound comprising a structureas in formula (IV):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino;

R₂=H, alkyl, or substituted alkyl;

Y=N or C;

R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, or combined withR₄ to form a carbocycle or heterocycle with the carbon atoms of thepyridine ring to which they are connected if Y=C;

R₄ is H, or substituted or unsubstituted alkyl, alkoxy, alkylthio, oralkylamino when not combined with R₃ to form a carbocycle or heterocyclewith the carbon atoms of the pyridine ring to which they are connected;or is combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected;

R₅ is a polymer or unit thereof;

X is C, N, O, or S;

R₆ and R₇ are each independently H or substituted; and

R₉, R₁₀, R₁₁, and R₁₂ are each independently H, a halogen, OH, thio,NH₂, or substituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy,aryloxy, alkylthio, arylthio, alkylamino, or arylamino; and

heating the alcohol, catalyst, and compound.

In other embodiments, a method for making a conjugate that comprises astructure as in formula (II):

comprises:

combining an alcohol and a compound comprising a structure as in formula(IV):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino;

R₂=H, alkyl, or substituted alkyl;

Y=N or C;

R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, or combined withR₄ to form a carbocycle or heterocycle with the carbon atoms of thepyridine ring to which they are connected if Y=C;

R₄ is H, or substituted or unsubstituted alkyl, alkoxy, alkylthio, oralkylamino when not combined with R₃ to form a carbocycle or heterocyclewith the carbon atoms of the pyridine ring to which they are connected;or is combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected;

R₅ is a polymer or unit thereof;

X is C, N, O, or S;

R₆ and R₇ are each independently H or substituted; and

R₉, R₁₀, R₁₁, and R₁₂ are each independently H, a halogen, OH, thio,NH₂, or substituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy,aryloxy, alkylthio, arylthio, alkylamino, or arylamino;

heating the alcohol and compound; and

adding a catalyst.

In yet other embodiments, a method for making a conjugate that comprisesa structure as in formula (II):

comprises:

combining an alcohol, a catalyst, and a compound comprising a structureas in formula (IV):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino;

R₂=H, alkyl, or substituted alkyl;

Y=N or C;

R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, or combined withR₄ to form a carbocycle or heterocycle with the carbon atoms of thepyridine ring to which they are connected if Y=C;

R₄ is H, or substituted or unsubstituted alkyl, alkoxy, alkylthio, oralkylamino when not combined with R₃ to form a carbocycle or heterocyclewith the carbon atoms of the pyridine ring to which they are connected;or is combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected;

R₅ is a polymer or unit thereof;

X is C, N, O, or S;

R₆ and R₇ are each independently H or substituted; and

R₉, R₁₀, R₁₁, and R₁₂ are each independently H, a halogen, OH, thio,NH₂, or substituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy,aryloxy, alkylthio, arylthio, alkylamino, or arylamino.

Imides that may be used in the aforementioned reactions are alsoprovided herein. In one embodiment, the imide compound comprises astructure as in formula (IV):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino;

R₂=H, alkyl, or substituted alkyl;

Y=N or C;

R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, or combined withR₄ to form a carbocycle or heterocycle with the carbon atoms of thepyridine ring to which they are connected if Y=C;

R₄ is H, or substituted or unsubstituted alkyl, alkoxy, alkylthio, oralkylamino when not combined with R₃ to form a carbocycle or heterocyclewith the carbon atoms of the pyridine ring to which they are connected;or is combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected;

X is C, N, O, or S;

R₆ and R₇ are each independently H or substituted; and

R₉, R₁₀, R₁₁, and R₁₂ are each independently H, a halogen, OH, thio,NH₂, or substituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy,aryloxy, alkylthio, arylthio, alkylamino, or arylamino.

Such a compound can be made by methods that comprise combining, in thepresence of a solvent and/or heat, with or without a dehydrating agent,such as a carboxylic acid anhydride or acetic anhydride, and a base,such as pyridine compound, a compound that comprises a structure as informula (III):

and

a compound comprising a structure as in formula (V):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino;

R₂=H, alkyl, or substituted alkyl;

Y=N or C;

R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, or combined withR₄ to form a carbocycle or heterocycle with the carbon atoms of thepyridine ring to which they are connected if Y=C;

R₄ is H, or substituted or unsubstituted alkyl, alkoxy, alkylthio, oralkylamino when not combined with R₃ to form a carbocycle or heterocyclewith the carbon atoms of the pyridine ring to which they are connected;or is combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected;

X is C, N, O, or S;

R₆ and R₇ are each independently H or substituted; and

R₉, R₁₀, R₁₁, and R₁₂ are each independently H, a halogen, OH, thio,NH₂, or substituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy,aryloxy, alkylthio, arylthio, alkylamino, or arylamino.

The inventors have also unexpectedly and surprisingly discovered that itis possible to make polymeric synthetic nanocarriers using polymers thathave a weight average molecular weight ranging from about 800 Daltons toabout 10,000 Daltons, as determined using gel permeation chromatography.In the formulation of polymeric synthetic nanocarriers, it has beengenerally believed that the molecular weight of polymers should be orexceed 10,000 Daltons. At times, it is advantageous to append to thepolymers an immunomodulatory compound that can be released from thesynthetic nanocarrier by a nonspecific degradation step within the body.If the synthetic nanocarriers are to be used to target theendosomal/lysosomal compartment, then it is particularly advantageous tohave this degradation step occur preferentially at an acidic pH. Onedrawback to appending the immunomodulatory agent to the polymer is thatthe loading is diminished as the molecular weight of the polymerincreases. In addition, as the molecular weight of the polymer increasesso does the hydrophobicity of the polymer with the results that thedegradation rate at a given pH can decrease. This leads to anundesirably decreased release rate of the immunomodulatory agent.Surprisingly, it has been found that low molecular weight polymers witha weight average molecular weight ranging from about 800 Daltons toabout 10,000 Daltons form stable synthetic nanocarriers and that therate of release of immunomodulatory agent from the synthetic nanocarrieris increased as the molecular weight decreases. The polymer of thecompounds provided herein, therefore, in embodiments, have a weightaverage molecular weight ranging from about 800 Daltons to about 10,000Daltons, and such compounds may be used to produce syntheticnanocarriers.

The compounds provided herein or the synthetic nanocarriers thatcomprise the compounds may also be pH sensitive (i.e., exhibit increasedrelease of the immunomodulatory agent at or about a pH of 4.5 ascompared to the release of the immunomodulatory agent at or aboutphysiological pH (i.e., pH or 7.4). The property of having relativelylow release of immunomodulatory agents at or about physiological pH butincreased release at or about a pH of 4.5 is desirable for it targetsthe immunomodulatory agents to the endosomal/lysosomal compartment of,for example, antigen presenting cells (APCs) which tend to possess a pHthat is at or about 4.5. This low pH level is found primarily in theupper gastrointestinal tract and endosome/lysosomes. Accordingly, unlessthe inventive compounds and compositions are administered via an oralroute of administration, accelerated release at pH at or about 4.5provides for an enhanced concentration of the immunomodulatory agent inthe target compartment. Under these conditions, the immunomodulatoryagent exhibits a pH sensitive dissociation and is then free to interactwith receptors within the endosome/lysosome and stimulate a desiredimmune response. Additionally, because the coupling of the polymer mayoccur at a position on the immunomodulatory agent or compound ofinterest that, generally, substantially reduces or eliminates thebiological activity of the immunomodulatory agent or compound ofinterest, the coupling can effectively produce a “pro-drug” like effect.This effect, in combination with accelerated release in conditionspresent in the endosome/lysosome, means that off-target effects (e.g.,adverse events) are reduced and safety margins increased forcompositions and vaccines that comprise the inventive compounds andcompositions.

The present invention will now be described in more detail.

Definitions

“Administering” or “administration” means providing a compound,conjugate, synthetic nanocarrier, or composition provided herein to apatient in a manner that is pharmacologically useful.

“APC targeting feature” means one or more portions of which theinventive synthetic nanocarriers are comprised that target the syntheticnanocarriers to professional antigen presenting cells (“APCs”), such asbut not limited to dendritic cells, SCS macrophages, folliculardendritic cells, and B cells. In embodiments, APC targeting features maycomprise immunofeature surface(s) and/or targeting moieties that bindknown targets on APCs. In embodiments, APC targeting features maycomprise one or more B cell antigens present on a surface of syntheticnanocarriers. In embodiments, APC targeting features may also compriseone or more dimensions of the synthetic nanoparticles that is selectedto promote uptake by APCs.

In embodiments, targeting moieties for known targets on macrophages(“Mphs”) comprise any targeting moiety that specifically binds to anyentity (e.g., protein, lipid, carbohydrate, small molecule, etc.) thatis prominently expressed and/or present on macrophages (i.e.,subcapsular sinus-Mph markers). Exemplary SCS-Mph markers include, butare not limited to, CD4 (L3T4, W3/25, T4); CD9 (p24, DRAP-1, MRP-1);CD11a (LFA-1α, α L Integrin chain); CD11b (αM Integrin chain, CR3, Mo1,C3niR, Mac-1); CD11c (αX Integrin, p150, 95, AXb2); CDw12 (p90-120);CD13 (APN, gp150, EC 3.4.11.2); CD14 (LPS-R); CD15 (X-Hapten, Lewis, X,SSEA-1, 3-FAL); CD15s (Sialyl Lewis X); CD15u (3′ sulpho Lewis X);CD15su (6 sulpho-sialyl Lewis X); CD16a (FCRIIIA); CD16b (FcgRIIIb);CDw17 (Lactosylceramide, LacCer); CD18 (Integrin β2, CD11a,b,cβ-subunit); CD26 (DPP IV ectoeneyme, ADA binding protein); CD29(Platelet GPIIa, β-1 integrin, GP); CD31 (PECAM-1, Endocam); CD32(FCγRII); CD33 (gp67); CD35 (CR1, C3b/C4b receptor); CD36 (GpIIIb, GPIV,PASIV); CD37 (gp52-40); CD38 (ADP-ribosyl cyclase, T10); CD39(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD43 (Sialophorin,Leukosialin); CD44 (EMCRII, H-CAM, Pgp-1); CD45 (LCA, T200, B220, Ly5);CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP,OA3, Neurophillin); CD47R (MEM-133); CD48 (Blast-1, Hulym3, BCM-1,OX-45); CD49a (VLA-1α, α1 Integrin); CD49b (VLA-2α, gpla, α2 Integrin);CD49c (VLA-3α, α3 Integrin); CD49e (VLA-5α, α5 Integrin); CD49f (VLA-6α,α6 Integrin, gplc); LD50 (ICAM-3); CD51 (Integrin α, VNR-α,Vitronectin-Rα); CD52 (CAMPATH-1, HE5); CD53 (OX-44); CD54 (ICAM-1);CD55 (DAF); CD58 (LFA-3); CD59 (1F5Ag, H19, Protectin, MACIF, MIRL,P-18); CD60a (GD3); CD60b (9-O-acetyl GD3); CD61 (GP IIIa, β3 Integrin);CD62L (L-selectin, LAM-1, LECAM-1, MEL-14, Leu8, TQ1); CD63 (LIMP, MLA1,gp55, NGA, LAMP-3, ME491); CD64 (FcγRI); CD65 (Ceramide, VIM-2); CD65s(Sialylated-CD65, VIM2); CD72 (Ly-19.2, Ly-32.2, Lyb-2); CD74 (Ii,invariant chain); CD75 (sialo-masked Lactosamine); CD75S (α2,6sialylated Lactosamine); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD82 (4F9,C33, IA4, KAI1, R2); CD84 (p75, GR6); CD85a (ILT5, LIR2, HL9); CD85d(ILT4, LIR2, MIR10); CD85j (ILT2, LIR1, MIR7); CD85k (ILT3, LIR5, HM18);CD86 (B7-2/B70); CD87 (uPAR); CD88 (C5aR); CD89 (IgA Fc receptor, FcαR);CD91 (α2M-R, LRP); CDw92 (p70); CDw93 (GR11); CD95 (APO-1, FAS,TNFRSF6); CD97 (BL-KDD/F12); CD98 (4F2, FRP-1, RL-388); CD99 (MIC2, E2);CD99R (CD99 Mab restricted); CD100 (SEMA4D); CD101 (IGSF2, P126, V7);CD102 (ICAM-2); CD111 (PVRL1, HveC, PRR1, Nectin 1, HIgR); CD112 (HveB,PRR2, PVRL2, Nectin2); CD114 (CSF3R, G-CSRF, HG-CSFR); CD115 (c-fms,CSF-1R, M-CSFR); CD116 (GMCSFRα); CDw119 (IFNγR, IFNγRA); CD120a (TNFRI,p55); CD120b (TNFRII, p75, TNFR p80); CD121b (Type 2 IL-1R); CD122(IL2Rβ); CD123 (IL-3Rα); CD124 (IL-4Rα); CD127 (p90, IL-7R, IL-7Rα);CD128a (IL-8Ra, CXCR1, (Tentatively renamed as CD181)); CD128b (IL-8Rb,CSCR2, (Tentatively renamed as CD182)); CD130 (gp130); CD131 (Common βsubunit); CD132 (Common γ chain, IL-2Rγ); CDw136 (MSP-R, RON, p158-ron);CDw137 (4-1BB, ILA); CD139; CD141 (Thrombomodulin, Fetomodulin); CD147(Basigin, EMMPRIN, M6, OX47); CD148 (HPTP-η, p260, DEP-1); CD155 (PVR);CD156a (CD156, ADAM8, MS2); CD156b (TACE, ADAM17, cSVP); CDw156C(ADAM10); CD157 (Mo5, BST-1); CD162 (PSGL-1); CD164 (MGC-24, MUC-24);CD165 (AD2, gp37); CD168 (RHAMM, IHABP, HMMR); CD169 (Sialoadhesin,Siglec-1); CD170 (Siglec 5); CD171 (L1CAM, NILE); CD172 (SIRP-1α,MyD-1); CD172b (SIRPβ); CD180 (RP105, Bgp95, Ly64); CD181 (CXCR1,(Formerly known as CD128a)); CD182 (CXCR2, (Formerly known as CD128b));CD184 (CXCR4, NPY3R); CD191 (CCR1); CD192 (CCR2); CD195 (CCR5); CDw197(CCR7 (was CDw197)); CDw198 (CCR8); CD204 (MSR); CD205 (DEC-25); CD206(MMR); CD207 (Langerin); CDw210 (CK); CD213a (CK); CDw217 (CK); CD220(Insulin R); CD221 (IGF1R); CD222 (M6P-R, IGFII-R); CD224 (GGT); CD226(DNAM-1, PTA1); CD230 (Prion Protein (PrP)); CD232 (VESP-R); CD244 (2B4,P38, NAIL); CD245 (p220/240); CD256 (APRIL, TALL2, TNF (ligand)superfamily, member 13); CD257 (BLYS, TALL1, TNF (ligand) superfamily,member 13b); CD261 (TRAIL-R1, TNF-R superfamily, member 10a); CD262(TRAIL-R2, TNF-R superfamily, member 10b); CD263 (TRAIL-R3, TNBF-Rsuperfamily, member 10c); CD264 (TRAIL-R4, TNF-R superfamily, member10d); CD265 (TRANCE-R, TNF-R superfamily, member 11a); CD277 (BT3.1, B7family: Butyrophilin 3); CD280 (TEM22, ENDO180); CD281 (TLR1, TOLL-likereceptor 1); CD282 (TLR2, TOLL-like receptor 2); CD284 (TLR4, TOLL-likereceptor 4); CD295 (LEPR); CD298 (ATP1B3, Na K ATPase, β3 subunit);CD300a (CMRF-35H); CD300c (CMRF-35A); CD300e (CMRF-35L1); CD302 (DCL1);CD305 (LAIR1); CD312 (EMR2); CD315 (CD9P1); CD317 (BST2); CD321 (JAM1);CD322 (JAM2); CDw328 (Siglec7); CDw329 (Siglec9); CD68 (gp 110,Macrosialin); and/or mannose receptor; wherein the names listed inparentheses represent alternative names.

In embodiments, targeting moieties for known targets on dendritic cells(“DCs”) comprise any targeting moiety that specifically binds to anyentity (e.g., protein, lipid, carbohydrate, small molecule, etc.) thatis prominently expressed and/or present on DCs (i.e., a DC marker).Exemplary DC markers include, but are not limited to, CD1a (R4, T6,HTA-1); CD1b (R1); CD1c (M241, R7); CD1d (R3); CD1e (R2); CD11b (αMIntegrin chain, CR3, Mo1, C3niR, Mac-1); CD11c (αX Integrin, p150, 95,AXb2); CDw117 (Lactosylceramide, LacCer); CD19 (B4); CD33 (gp67); CD 35(CR1, C3b/C4b receptor); CD 36 (GpIIIb, GPIV, PASIV); CD39(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD45 (LCA, T200,B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD49d (VLA-4α, α4Integrin); CD49e (VLA-5α, α5 Integrin); CD58 (LFA-3); CD64 (FcγRI); CD72(Ly-19.2, Ly-32.2, Lyb-2); CD73 (Ecto-5′nucloticlase); CD74 (Ii,invariant chain); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD83 (HB15);CD85a (ILT5, LIR3, HL9); CD85d (ILT4, LIR2, MIR10); CD85j (ILT2, LIR1,MIR7); CD85k (ILT3, LIR5, HM18); CD86 (B7-2/B70); CD88 (C5aB); CD97(BL-KDD/F12); CD101 (IGSF2, P126, V7); CD116 (GM-CSFRα); CD120a (TMFRI,p55); CD120b (TNFRII, p75, TNFR p80); CD123 (IL-3Ra); CD139; CD148(HPTP-η, DEP-1); CD150 (SLAM, IPO-3); CD156b (TACE, ADAM17, cSVP); CD157(Mo5, BST-1); CD167a (DDR1, trkE, cak); CD168 (RHAMM, IHABP, HMMR);CD169 (Sialoadhesin, Siglec-1); CD170 (Siglec-5); CD171 (L1CAM, NILE);CD172 (SIRP-1α, MyD-1); CD172b (SIRPβ); CD180 (RP105, Bgp95, Ly64);CD184 (CXCR4, NPY3R); CD193 (CCR3); CD196 (CCR6); CD197 (CCR7 (wsCDw197)); CDw197 (CCR7, EBI1, BLR2); CD200 (OX2); CD205 (DEC-205); CD206(MMR); CD207 (Langerin); CD208 (DC-LAMP); CD209 (DCSIGN); CDw218a(IL18Rα); CDw218b (IL8Rβ); CD227 (MUC1, PUM, PEM, EMA); CD230 (PrionProtein (PrP)); CD252 (OX40L, TNF (ligand) superfamily, member 4); CD258(LIGHT, TNF (ligand) superfamily, member 14); CD265 (TRANCE-R, TNF-Rsuperfamily, member 11a); CD271 (NGFR, p75, TNFR superfamily, member16); CD273 (B7DC, PDL2); CD274 (B7H1, PDL1); CD275 (B7H2, ICOSL); CD276(B7H3); CD277 (BT3.1, B7 family: Butyrophilin 3); CD283 (TLR3, TOLL-likereceptor 3); CD289 (TLR9, TOLL-like receptor 9); CD295 (LEPR); CD298(ATP1B3, Na K ATPase β3 submit); CD300a (CMRF-35H); CD300c (CMRF-35A);CD301 (MGL1, CLECSF14); CD302 (DCL1); CD303 (BDCA2); CD304 (BDCA4);CD312 (EMR2); CD317 (BST2); CD319 (CRACC, SLAMF7); CD320 (8D6); and CD68(gp110, Macrosialin); class II MHC; BDCA-1; Siglec-H; wherein the nameslisted in parentheses represent alternative names.

In embodiments, targeting can be accomplished by any targeting moietythat specifically binds to any entity (e.g., protein, lipid,carbohydrate, small molecule, etc.) that is prominently expressed and/orpresent on B cells (i.e., B cell marker). Exemplary B cell markersinclude, but are not limited to, CD1c (M241, R7); CD1d (R3); CD2(E-rosette R, T11, LFA-2); CD5 (T1, Tp67, Leu-1, Ly-1); CD6 (T12); CD9(p24, DRAP-1, MRP-1); CD11a (LFA-1α, αL Integrin chain); CD11b (αMIntegrin chain, CR3, Mo1, C3niR, Mac-1); CD11c (αX Integrin, P150, 95,AXb2); CDw17 (Lactosylceramide, LacCer); CD18 (Integrin β2, CD11a, b, cβ-subunit); CD19 (B4); CD20 (B1, Bp35); CD21 (CR2, EBV-R, C3dR); CD22(BL-CAM, Lyb8, Siglec-2); CD23 (FceRII, B6, BLAST-2, Leu-20); CD24(BBA-1, HSA); CD25 (Tac antigen, IL-2Ra, p55); CD26 (DPP IV ectoeneyme,ADA binding protein); CD27 (T14, S152); CD29 (Platelet GPIIa, β-1integrin, GP); CD31 (PECAM-1, Endocam); CD32 (FCγRII); CD35 (CR1,C3b/C4b receptor); CD37 (gp52-40); CD38 (ADPribosyl cyclase, T10); CD39(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD44 (ECMRII,H-CAM, Pgp-1); CD45 (LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC;CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP, OA3, Neurophilin); CD47R(MEM-133); CD48 (Blast-1, Hulym3, BCM-1, OX-45); CD49b (VLA-2α, gpla, α2Integrin); CD49c (VLA-3α, α3 Integrin); CD49d (VLA-4α, α4 Integrin);LD50 (ICAM-3); CD52 (CAMPATH-1, HES); CD53 (OX-44); CD54 (ICAM-1); CD55(DAF); CD58 (LFA-3); CD60a (GD3); CD62L (L-selectin, LAM-1, LECAM-1,MEL-14, Leu8, TQ1); CD72 (Ly-19.2, Ly-32.2, Lyb-2); CD73(Ecto-5′-nuciotidase); CD74 (Ii, invariant chain); CD75 (sialo-maskedLactosamine); CD75S (α2, 6 sialytated Lactosamine); CD77 (Pk antigen,BLA, CTH/Gb3); CD79a (Igα, MB1); CD79b (Igβ, B29); CD80; CD81 (TAPA-1);CD82 (4F9, C33, IA4, KAI1, R2); CD83 (HB15); CD84 (P75, GR6); CD85j(ILT2, LIR1, MIR7); CDw92 (p70); CD95 (APO-1, FAS, TNFRSF6); CD98 (4F2,FRP-1, RL-388); CD99 (MIC2, E2); CD100 (SEMA4D); CD102 (ICAM-2); CD108(SEMA7A, JMH blood group antigen); CDw119 (IFNγR, IFNγRa); CD120a(TNFRI, p55); CD120b (TNFRII, p75, TNFR p80); CD121b (Type 2 IL-1R);CD122 (IL2Rβ); CD124 (IL-4Rα); CD130 (gp130); CD132 (Common γ chain,IL-2Rγ); CDw137 (4-1BB, ILA); CD139; CD147 (Basigin, EMMPRIN, M6, OX47);CD150 (SLAM, IPO-3); CD162 (PSGL-1); CD164 (MGC-24, MUC-24); CD166(ALCAM, KG-CAM, SC-1, BEN, DM-GRASP); CD167a (DDR1, trkE, cak); CD171(L1CMA, NILE); CD175s (Sialyl-Tn (S-Tn)); CD180 (RP105, Bgp95, Ly64);CD184 (CXCR4, NPY3R); CD185 (CXCR5); CD192 (CCR2); CD196 (CCR6); CD197(CCR7 (was CDw197)); CDw197 (CCR7, EBI1, BLR2); CD200 (OX2); CD205(DEC-205); CDw210 (CK); CD213a (CK); CDw217 (CK); CDw218a (IL18Rα);CDw218b (IL18Rβ); CD220 (Insulin R); CD221 (IGF1R); CD222 (M6P-R,IGFII-R); CD224 (GGT); CD225 (Leu13); CD226 (DNAM-1, PTA1); CD227 (MUC1,PUM, PEM, EMA); CD229 (Ly9); CD230 (Prion Protein (Prp)); CD232(VESP-R); CD245 (p220/240); CD24? (CD3 Zeta Chain); CD261 (TRAIL-R1,TNF-R superfamily, member 10a); CD262 (TRAIL-R2, TNF-R superfamily,member 10b); CD263 (TRAIL-R3, TNF-R superfamily, member 10c); CD264(TRAIL-R4, TNF-R superfamily, member 10d); CD265 (TRANCE-R, TNF-Rsuperfamily, member 11a); CD267 (TACI, TNF-R superfamily, member 13B);CD268 (BAFFR, TNF-R superfamily, member 13C); CD269 (BCMA, TNF-Rsuperfamily, member 16); CD275 (B7H2, ICOSL); CD277 (BT3.1.B7 family:Butyrophilin 3); CD295 (LEPR); CD298 (ATP1B3 Na K ATPase β3 subunit);CD300a (CMRF-35H); CD300c (CMRF-35A); CD305 (LAIR1); CD307 (IRTA2);CD315 (CD9P1); CD316 (EW12); CD317 (BST2); CD319 (CRACC, SLAMF7); CD321(JAM1); CD322 (JAM2); CDw327 (Siglec6, CD33L); CD68 (gp 100,Macrosialin); CXCR5; VLA-4; class II MHC; surface IgM; surface IgD;APRL; and/or BAFF-R; wherein the names listed in parentheses representalternative names. Examples of markers include those provided elsewhereherein.

In some embodiments, B cell targeting can be accomplished by anytargeting moiety that specifically binds to any entity (e.g., protein,lipid, carbohydrate, small molecule, etc.) that is prominently expressedand/or present on B cells upon activation (i.e., activated B cellmarker). Exemplary activated B cell markers include, but are not limitedto, CD1a (R4, T6, HTA-1); CD1b (R1); CD15s (Sialyl Lewis X); CD15u (3′sulpho Lewis X); CD15su (6 sulpho-sialyl Lewis X); CD30 (Ber-H2, Ki-1);CD69 (AIM, EA 1, MLR3, gp34/28, VEA); CD70 (Ki-24, CD27 ligand); CD80(B7, B7-1, BB1); CD86 (B7-2/B70); CD97 (BLKDD/F12); CD125 (IL-5Rα);CD126 (IL-6Rα); CD138 (Syndecan-1, Heparan sulfate proteoglycan); CD152(CTLA-4); CD252 (OX40L, TNF(ligand) superfamily, member 4); CD253(TRAIL, TNF(ligand) superfamily, member 10); CD279 (PD1); CD289 (TLR9,TOLL-like receptor 9); and CD312 (EMR2); wherein the names listed inparentheses represent alternative names. Examples of markers includethose provided elsewhere herein.

“B cell antigen” means any antigen that naturally is or could beengineered to be recognized by a B cell, and triggers (naturally orbeing engineered as known in the art) an immune response in a B cell(e.g., an antigen that is specifically recognized by a B cell receptoron a B cell). In some embodiments, an antigen that is a T cell antigenis also a B cell antigen. In other embodiments, the T cell antigen isnot also a B cell antigen. B cell antigens include, but are not limitedto proteins, peptides, small molecules, and carbohydrates. In someembodiments, the B cell antigen is a non-protein antigen (i.e., not aprotein or peptide antigen). In some embodiments, the B cell antigen isa carbohydrate associated with an infectious agent. In some embodiments,the B cell antigen is a glycoprotein or glycopeptide associated with aninfectious agent. The infectious agent can be a bacterium, virus,fungus, protozoan, parasite or prion. In some embodiments, the B cellantigen is a poorly immunogenic antigen. In some embodiments, the B cellantigen is an abused substance or a portion thereof. In someembodiments, the B cell antigen is an addictive substance or a portionthereof. Addictive substances include, but are not limited to, nicotine,a narcotic, a cough suppressant, a tranquilizer, and a sedative. In someembodiments, the B cell antigen is a toxin, such as a toxin from achemical weapon or natural sources, or a pollutant. The B cell antigenmay also be a hazardous environmental agent. In other embodiments, the Bcell antigen is an alloantigen, an allergen, a contact sensitizer, adegenerative disease antigen, a hapten, an infectious disease antigen, acancer antigen, an atopic disease antigen, an addictive substance, axenoantigen, or a metabolic disease enzyme or enzymatic product thereof.

“Biodegradable polymer” means a polymer that degrades over time whenintroduced into the body of a subject. Biodegradable polymers, includebut are not limited to, polyesters, polycarbonates, polyketals, orpolyamides. Such polymers may comprise poly(lactic acid), poly(glycolicacid), poly(lactic-co-glycolic acid), or polycaprolactone. In someembodiments, the biodegradable polymer comprises a block-co-polymer of apolyether, such as poly(ethylene glycol), and a polyester,polycarbonate, or polyamide, or other biodegradable polymer. Inembodiments, the biodegradable polymer comprises a block-co-polymer ofpoly(ethylene glycol) and poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid), or polycaprolactone. In some embodiments,however, the biodegradable polymer does not comprise a polyether, suchas poly(ethylene glycol), or consist solely of the polyether. Generally,for use as part of a synthetic nanocarrier the biodegradable polymer isinsoluble in water at pH=7.4 and at 25° C. The biodegradable polymer, inembodiments, have a weight average molecular weight ranging from about800 to about 50,000 Daltons, as determined using gel permeationchromatography. In some embodiments, the weight average molecular weightis from about 800 Daltons to about 10,000 Daltons, preferably from 800Daltons to 10,000 Daltons, as determined using gel permeationchromatography. In other embodiments, the weight average molecularweight is from 1000 Daltons to 10,000 Daltons, as determined by gelpermeation chromatography. In an embodiment, the biodegradable polymerdoes not comprise polyketal or a unit thereof.

“Couple” or “Coupled” or “Couples” (and the like) means attached to apolymer or unit thereof or attached to or contained within the syntheticnanocarrier. In some embodiments, the covalent coupling is mediated byone or more linkers. In some embodiments, the coupling is non-covalent.In some embodiments, the non-covalent coupling is mediated by chargeinteractions, affinity interactions, metal coordination, physicaladsorption, hostguest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. In embodiments,the coupling may arise in the context of encapsulation within thesynthetic nanocarriers, using conventional techniques. Any of theaforementioned couplings may be arranged to be on a surface or within aninventive synthetic nanocarrier.

“Dosage form” means a compound, conjugate, synthetic nanocarrier, orcomposition provided herein in a medium, carrier, vehicle, or devicesuitable for administration to a subject.

“Encapsulate” means to enclose within a synthetic nanocarrier,preferably enclose completely within a synthetic nanocarrier. Most orall of a substance that is encapsulated is not exposed to the localenvironment external to the synthetic nanocarrier. Encapsulation isdistinct from absorbtion, which places most or all of a substance on asurface of a synthetic nanocarrier, and leaves the substance exposed tothe local environment external to the synthetic nanocarrier. Inembodiments, the immunomodulatory agent or B cell and/or T cell antigenis encapsulated within the synthetic nanocarrier.

“Immunomodulatory agent” means an agent that modulates an immuneresponse. “Modulate”, as used herein, refers to inducing, enhancing,stimulating, or directing an immune response. Such agents includeimmunostimulatory agents that stimulate (or boost) an immune response toan antigen but is not an antigen or derived from an antigen. In someembodiments, the immunomodulatory agent is on the surface of thesynthetic nanocarrier and/or is incorporated within the syntheticnanocarrier. In embodiments, the immunomodulatory agent is coupled tothe synthetic nanocarrier via the polymer or unit thereof of thecompounds or conjugates provided.

In some embodiments, all of the immunomodulatory agents of a syntheticnanocarrier are identical to one another. In some embodiments, asynthetic nanocarrier comprises a number of different types ofimmunomodulatory agents. In some embodiments, a synthetic nanocarriercomprises multiple individual immunomodulatory agents, all of which areidentical to one another. In some embodiments, a synthetic nanocarriercomprises exactly one type of immunomodulatory agent. In someembodiments, a synthetic nanocarrier comprises exactly two distincttypes of immunomodulatory agents. In some embodiments, a syntheticnanocarrier comprises greater than two distinct types ofimmunomodulatory agents.

“Maximum dimension of a synthetic nanocarrier” means the largestdimension of a nanocarrier measured along any axis of the syntheticnanocarrier. “Minimum dimension of a synthetic nanocarrier” means thesmallest dimension of a synthetic nanocarrier measured along any axis ofthe synthetic nanocarrier. For example, for a spheroidal syntheticnanocarrier, the maximum and minimum dimension of a syntheticnanocarrier would be substantially identical, and would be the size ofits diameter. Similarly, for a cubic synthetic nanocarrier, the minimumdimension of a synthetic nanocarrier would be the smallest of itsheight, width or length, while the maximum dimension of a syntheticnanocarrier would be the largest of its height, width or length. In anembodiment, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is greater than 100 nm. In an embodiment, a maximum dimension ofat least 75%, preferably at least 80%, more preferably at least 90%, ofthe synthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample, is equal to or less than 5 μm.Preferably, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is equal to or greater than 110 nm, more preferably equal to orgreater than 120 nm, more preferably equal to or greater than 130 nm,and more preferably still equal to or greater than 150 nm. Preferably, amaximum dimension of at least 75%, preferably at least 80%, morepreferably at least 90%, of the synthetic nanocarriers in a sample,based on the total number of synthetic nanocarriers in the sample isequal to or less than 3 μm, more preferably equal to or less than 2 μm,more preferably equal to or less than 1 μm, more preferably equal to orless than 800 nm, more preferably equal to or less than 600 nm, and morepreferably still equal to or less than 500 nm. In preferred embodiments,a maximum dimension of at least 75%, preferably at least 80%, morepreferably at least 90%, of the synthetic nanocarriers in a sample,based on the total number of synthetic nanocarriers in the sample, isequal to or greater than 100 nm, more preferably equal to or greaterthan 120 nm, more preferably equal to or greater than 130 nm, morepreferably equal to or greater than 140 nm, and more preferably stillequal to or greater than 150 nm. Measurement of synthetic nanocarriersizes is obtained by suspending the synthetic nanocarriers in a liquid(usually aqueous) media and using dynamic light scattering (e.g. using aBrookhaven ZetaPALS instrument).

“Pharmaceutically acceptable excipient” means a pharmacologicallyinactive substance added to an inventive compound, conjugate, syntheticnanocarrier or composition to further facilitate its administration.Examples, without limitation, of pharmaceutically acceptable excipientsinclude calcium carbonate, calcium phosphate, various diluents, varioussugars and types of starch, cellulose derivatives, gelatin, vegetableoils, and polyethylene glycols.

“Release Rate” means the rate that an entrapped immunomodulatory agentflows from a composition, such as a synthetic nanocarrier, into asurrounding media in an in vitro release test. First, the syntheticnanocarrier is prepared for the release testing by placing into theappropriate in vitro release media. This is generally done by exchangingthe buffer after centrifugation to pellet the synthetic nanocarrier andreconstitution of the synthetic nanocarriers using a mild condition. Theassay is started by placing the sample at 37° C. in an appropriatetemperature-controlled apparatus. A sample is removed at various timepoints.

The synthetic nanocarriers are separated from the release media bycentrifugation to pellet the synthetic nanocarriers. The release mediais assayed for the immunomodulatory agent that has dispersed from thesynthetic nanocarriers. The immunomodulatory agent is measured usingHPLC to determine the content and quality of the immunomodulatory agent.The pellet containing the remaining entrapped immunomodulatory agent isdissolved in solvents or hydrolyzed by base to free the entrappedimmunomodulatory agent from the synthetic nanocarriers. Thepellet-containing immunomodulatory agent is then also measured by HPLCto determine the content and quality of the immunomodulatory agent thathas not been released at a given time point.

The mass balance is closed between immunomodulatory agent that has beenreleased into the release media and what remains in the syntheticnanocarriers. Data are presented as the fraction released or as the netrelease presented as micrograms released over time.

“Subject” means an animal, including mammals such as humans andprimates; avians; domestic household or farm animals such as cats, dogs,sheep, goats, cattle, horses and pigs; laboratory animals such as mice,rats and guinea pigs; fish; and the like.

“Synthetic nanocarrier(s)” means a discrete object that is not found innature, and that possesses at least one dimension that is less than orequal to 5 microns in size. Albumin nanoparticles are expressly includedas synthetic nanocarriers.

Synthetic nanocarriers include the compounds and compositions providedherein and, therefore, can be polymeric nanoparticles. In someembodiments, synthetic nanocarriers can comprise one or more polymericmatrices. The synthetic nanocarriers, however, can also include othernanomaterials and may be, for example, lipid-polymer nanoparticles. Insome embodiments, a polymeric matrix can be surrounded by a coatinglayer (e.g., liposome, lipid monolayer, micelle, etc.). In someembodiments, the synthetic nanocarrier is not a micelle. In someembodiments, a synthetic nanocarrier may comprise a core comprising apolymeric matrix surrounded by a lipid layer (e.g., lipid bilayer, lipidmonolayer, etc.). In some embodiments, the various elements of thesynthetic nanocarriers can be coupled with the polymeric matrix.

The synthetic nanocarriers may comprise one or more lipids. In someembodiments, a synthetic nanocarrier may comprise a liposome. In someembodiments, a synthetic nanocarrier may comprise a lipid bilayer. Insome embodiments, a synthetic nanocarrier may comprise a lipidmonolayer. In some embodiments, a synthetic nanocarrier may comprise amicelle. In some embodiments, a synthetic nanocarrier may comprise anon-polymeric core (e.g., metal particle, quantum dot, ceramic particle,bone particle, viral particle, proteins, nucleic acids, carbohydrates,etc.) surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer,etc.).

The synthetic nanocarriers may comprise lipid-based nanoparticles,metallic nanoparticles, surfactant-based emulsions, dendrimers,buckyballs, nanowires, virus-like particles, peptide or protein-basedparticles (such as albumin nanoparticles). Synthetic nanocarriers may bea variety of different shapes, including but not limited to spheroidal,cubic, pyramidal, oblong, cylindrical, toroidal, and the like. Syntheticnanocarriers according to the invention comprise one or more surfaces.Exemplary synthetic nanocarriers that can be adapted for use in thepractice of the present invention comprise: (1) the biodegradablenanoparticles disclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2)the polymeric nanoparticles of Published U.S. Patent Application20060002852 to Saltzman et al., (3) the lithographically constructednanoparticles of Published U.S. Patent Application 20090028910 toDeSimone et al., (4) the disclosure of WO 2009/051837 to von Andrian etal., or (5) the nanoparticles disclosed in Published U.S. PatentApplication 2008/0145441 to Penades et al.

Synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface with hydroxyl groups thatactivate complement or alternatively comprise a surface that consistsessentially of moieties that are not hydroxyl groups that activatecomplement. In a preferred embodiment, synthetic nanocarriers accordingto the invention that have a minimum dimension of equal to or less thanabout 100 nm, preferably equal to or less than 100 nm, do not comprise asurface that substantially activates complement or alternativelycomprise a surface that consists essentially of moieties that do notsubstantially activate complement. In a more preferred embodiment,synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface that activates complement oralternatively comprise a surface that consists essentially of moietiesthat do not activate complement. In embodiments, synthetic nanocarriersmay possess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3,1:5, 1:7, or greater than 1:10.

In some embodiments, synthetic nanocarriers are spheres or spheroids. Insome embodiments, synthetic nanocarriers are flat or plate-shaped. Insome embodiments, synthetic nanocarriers are cubes or cubic. In someembodiments, synthetic nanocarriers are ovals or ellipses. In someembodiments, synthetic nanocarriers are cylinders, cones, or pyramids.

It is often desirable to use a population of synthetic nanocarriers thatis relatively uniform in terms of size, shape, and/or composition sothat each synthetic nanocarrier has similar properties. For example, atleast 80%, at least 90%, or at least 95% of the synthetic nanocarriersmay have a minimum dimension or maximum dimension that falls within 5%,10%, or 20% of the average diameter or average dimension. In someembodiments, a population of synthetic nanocarriers may be heterogeneouswith respect to size, shape, and/or composition.

Synthetic nanocarriers can be solid or hollow and can comprise one ormore layers. In some embodiments, each layer has a unique compositionand unique properties relative to the other layer(s). To give but oneexample, synthetic nanocarriers may have a core/shell structure, whereinthe core is one layer (e.g., a polymeric core) and the shell is a secondlayer (e.g., a lipid bilayer or monolayer). Synthetic nanocarriers maycomprise a plurality of different layers.

“T cell antigen” means any antigen that is recognized by and triggers animmune response in a T cell (e.g., an antigen that is specificallyrecognized by a T cell receptor on a T cell or an NKT cell viapresentation of the antigen or portion thereof bound to a Class I orClass II major histocompatability complex molecule (MHC), or bound to aCD1 complex). In some embodiments, an antigen that is a T cell antigenis also a B cell antigen. In other embodiments, the T cell antigen isnot also a B cell antigen. T cell antigens generally are proteins orpeptides. T cell antigens may be an antigen that stimulates a CD8+ Tcell response, a CD4+ T cell response, or both. The T cell antigens,therefore, in some embodiments can effectively stimulate both types ofresponses.

In some embodiments the T cell antigen is a T-helper antigen, which is aT cell antigen that can generate an augmented response to an unrelated Bcell antigen through stimulation of T cell help. In embodiments, aT-helper antigen may comprise one or more peptides derived from tetanustoxoid, Epstein-Barr virus, influenza virus, respiratory syncytialvirus, measles virus, mumps virus, rubella virus, cytomegalovirus,adenovirus, diphtheria toxoid, or a PADRE peptide. In other embodiments,a T-helper antigen may comprise one or more lipids, or glycolipids,including but not limited to: α-galactosylceramide (α-GalCer), α-linkedglycosphingolipids (from Sphingomonas spp.), galactosyl diacylglycerols(from Borrelia burgdorferi), lypophosphoglycan (from Leishmaniadonovani), and phosphatidylinositol tetramannoside (PIM4) (fromMycobacterium leprae). For additional lipids and/or glycolipids usefulas T-helper antigens, see V. Cerundolo et al., “Harnessing invariant NKTcells in vaccination strategies.” Nature Rev Immun, 9:28-38 (2009). Inembodiments, CD4+ T-cell antigens may be derivatives of a CD4+ T-cellantigen that is obtained from a source, such as a natural source. Insuch embodiments, CD4+ T-cell antigen sequences, such as those peptidesthat bind to MHC II, may have at least 70%, 80%, 90%, or 95% identity tothe antigen obtained from the source. In embodiments, the T cellantigen, preferably a T-helper antigen, may be coupled to, or uncoupledfrom, a synthetic nanocarrier.

“Unit thereof” refers to a monomeric unit of a polymer, the polymergenerally being made up of a series of linked monomers.

“Vaccine” means a composition of matter that improves the immuneresponse to a particular pathogen or disease. A vaccine typicallycontains factors that stimulate a subject's immune system to recognize aspecific antigen as foreign and eliminate it from the subject's body. Avaccine also establishes an immunologic ‘memory’ so the antigen will bequickly recognized and responded to if a person is re-challenged.Vaccines can be prophylactic (for example to prevent future infection byany pathogen), or therapeutic (for example a vaccine against a tumorspecific antigen for the treatment of cancer). Vaccines according to theinvention may comprise one or more of the compounds, conjugates,synthetic nanocarriers, or compositions provided herein.

Methods of Making the Inventive Compounds, Conjugates, or SyntheticNanocarriers

The immunomodulatory agent and polymers or unit thereof are coupledcovalently via an amide or ester bond. In some embodiments, theseconjugates form part of a synthetic nanocarrier. In general, a polymer,such as polylactide (PLA) or polylactide-co-glycolide (PLGA), can beconjugated with an immunostimulatory agent, such as resiquimod (alsoknown as R848), in several ways. Methods for coupling are provided belowand in the EXAMPLES.

The following methods or any step of the methods provided are exemplaryand may be carried out under any suitable conditions. In some cases, thereaction or any step of the methods provided may be carried out in thepresence of a solvent or a mixture of solvents. Non-limiting examples ofsolvents that may be suitable for use in the invention include, but arenot limited to, p-cresol, toluene, xylene, mesitylene, diethyl ether,glycol, petroleum ether, hexane, cyclohexane, pentane, dichloromethane(or methylene chloride), chloroform, dioxane, tetrahydrofuran (THF),dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate(EtOAc), triethylamine, acetonitrile, methyl-t-butyl ether (MTBE),N-methylpyrrolidone (NMP), dimethylacetamide (DMAC), isopropanol (IPA),mixtures thereof, or the like. In some cases, the solvent is selectedfrom the group consisting of ethyl acetate, methylene chloride, THF,DMF, NMP, DMAC, DMSO, and toluene, or a mixture thereof.

A reaction or any step of the methods provided may be carried out at anysuitable temperature. In some cases, a reaction or any step of themethods provided is carried out at about room temperature (e.g., about25° C., about 20° C., between about 20° C. and about 25° C., or thelike). In some cases, however, the reaction or any step of the methodsprovided may be carried out at a temperature below or above roomtemperature, for example, at about −20° C., at about −10° C., at about0° C., at about 10° C., at about 30° C., about 40° C., about 50° C.,about 60° C., about 70° C., about 80° C., about 90° C., about 100° C.,about 120° C., about 140° C., about 150° C. or greater. In particularembodiments, the reaction or any step of the methods provided isconducted at temperatures between 0° C. and 120° C. In some embodiments,the reaction or any step of the methods provided may be carried out atmore than one temperature (e.g., reactants added at a first temperatureand the reaction mixture agitated at a second wherein the transitionfrom a first temperature to a second temperature may be gradual orrapid).

The reaction or any step of the methods provided may be allowed toproceed for any suitable period of time. In some cases, the reaction orany step of the methods provided is allowed to proceed for about 10minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours,about 12 hours, about 16 hours, about 24 hours, about 2 days, about 3days, about 4 days, or more. In some cases, aliquots of the reactionmixture may be removed and analyzed at an intermediate time to determinethe progress of the reaction or any step of the methods provided. Insome embodiments, a reaction or any step of the methods provided may becarried out under an inert atmosphere in anhydrous conditions (e.g.,under an atmosphere of nitrogen or argon, anhydrous solvents, etc.)

The reaction products and/or intermediates may be isolated (e.g., viadistillation, column chromatography, extraction, precipitation, etc.)and/or analyzed (e.g., gas liquid chromatography, high performanceliquid chromatography, nuclear magnetic resonance spectroscopy, etc.)using commonly known techniques. In some cases, a conjugate or syntheticnanocarrier that includes the conjugated may be analyzed to determinethe loading of immunomodulatory agent, for example, using reverse phaseHPLC.

The polymers may have any suitable molecular weight. For example, thepolymers may have a low or high molecular weight. Non-limiting molecularweight values include 100 Da, 200 Da, 300 Da, 500 Da, 750 Da, 1000 Da,2000 Da, 3000 Da, 4000 Da, 5000 Da, 6000 Da, 7000 Da, 8000 Da, 9000 Da,10,000 Da, or greater. In some embodiments, the polymers have a weightaverage molecular weight of about 800 Da to about 10,000 Da. Themolecular weight of a polymer may be determined using gel permeationchromatography.

Provided below are exemplary conjugation reactions that are not intendedto be limiting.

Method 1

A polymer (e.g., PLA, PLGA) or unit thereof with at least one acid endgroups is converted to a reactive acylating agent such as an acylhalide, acylimidazole, active ester, etc. using an activating reagentcommonly used in amide synthesis.

In this two-step method, the resulting activated polymer or unit thereof(e.g., PLA, PLGA) is isolated and then reacted with an immunomodulatoryagent (e.g., R848) in the presence of a base to give the desiredconjugate (e.g., PLA-R848), for example, as shown in the followingscheme:

Activating reagents that can be used to convert polymers or unitsthereof, such as PLA or PLGA, to an activated acylating form include,but are not limited to cyanuric fluoride,N,N-tetramethylfluoroformamidinium hexafluorophosphate (TFFH);Acylimidazoles, such as carbonyl diimidazole (CDI),N,N′-carbonylbis(3-methylimidazolium) triflate (CBMIT); and Activeesters, such as N-hydroxylsuccinimide (NHS or HOSu) in the presence of acarbodiimide such as N,N′-dicyclohexylcarbodiimide (DCC),N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) orN, N′-diisopropylcarbodiimide (DIC); N,N′-disuccinimidyl carbonate(DSC); pentaflurophenol in the presence of DCC or EDC or DIC;pentafluorophenyl trifluoroacetate.

The activated polymer or unit thereof may be isolated (e.g., viaprecipitation, extraction, etc.) and/or stored under suitable conditions(e.g., at low temperature, under argon) following activation, or may beused immediately. The activated polymer or unit thereof may be reactedwith an immunostimulatory agent under any suitable conditions. In somecases, the reaction is carried out in the presence of a base and/orcatalyst. Non-limiting examples of bases/catalysts includediisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP).

Method 2

A polymer or unit thereof (e.g., PLA, PLGA having any suitable molecularweight) with an acid end group reacts with an immunomodulatory agent(e.g., R848) in the presence of an activating or coupling reagent, whichconverts the polymer or unit thereof (e.g., PLA, PLGA) to a reactiveacylating agent in situ, to give the desired conjugate (e.g., PLA-R848,PLGA-R848).

Coupling or activating agents include but are not limited to: activatingagents used in the presence of an carbodiimide such as EDC or DCC orDIC, such as 1-Hydroxybenzotriazole (HOBt), 1-Hydroxy-7-azabenzotriazole(HOAt), 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HO-Dhbt),N-Hydroxysuccinimide (NHS or HOSu), Pentafluorophenol (PFP); Activatingagents without carbodiimide: Phosphonium salts, such asO-Benzotriazol-1-yloxytris(dimethylamino) phosphoniumhexafluorophosphate (BOP),O-Benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate(PyBOP), 7-Azabenzotriazol-1-yloxytris(pyrrolidino)phosphoniumhexafluorophosphate (PyAOP); uronium salts such asO-Benzotriazol-1-yloxytris-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) and hexafluorophosphate (HBTU),O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),O-(1,2-dihydro-2-oxo-1-pyridyl)-1,1,3,3-tetramethyl-uroniumtetrafluoroborate (TPTU); Halouronium and halophosphonium salts such asbis(tetramethylene)fluoroformamidinium hexafluorophosphate (BTFFH),bromotris(dimethylamino) phosphonium hexafluorophosphate (BroP),bromotripyrrolidino phosphonium hexafluorophosphate (PyBroP) andchlorotripyrrolidino phosphonium hexafluorophosphate (PyClop);Benzotriazine derivatives such asO-(3,4-Dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TDBTU) and3-(diethyloxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT).Non-limiting examples of suitable solvents include DMF, DCM, toluene,ethyl acetate, etc., as described herein.

Method 3

Immunomodulatory agents, such as R848, can also be coupled to polymersor units thereof that are terminated in a hydroxyl group. Such polymersor units thereof include polyethylene glycol, polylactide,polylactide-co-glycolide, polycaprolactone, and other like polyesters,or units thereof. In general, the reaction proceeds as follows where animide of the general structure (IV) will react with the terminalhydroxyl of the aforementioned polymers or units thereof using acatalyst used in lactone ring opening polymerizations. The resultingreaction product (II) links the amide of the agent to the polymer orunit thereof via an ester bond. The compounds of formula (IV) and (II)are as follows:

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; R₅ is a polymeror unit thereof; X is C, N, O, or S; R₆ and R₇ are each independently Hor substituted; and R₉, R₁₀, R₁₁, and R₁₂ are each independently H, ahalogen, OH, thio, NH₂, or substituted or unsubstituted alkyl, aryl,heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, orarylamino.

Catalysts include, but are not limited to, phosphazine bases,1,8-diazabicycloundec-7-ene (DBU), 1,4,7-triazabicyclodecene (TBD), andN-methyl-1,4,7-triazabicyclodecene (MTDB). Other catalysts are known inthe art and provided, for example, in Kamber et al., OrganocatalyticRing-Opening Polymerization, Chem. Rev. 2007, 107, 58-13-5840.Non-limiting examples of suitable solvents include methylene chloride,chloroform, and THF.

A specific example of a reaction completed by such a method is shownhere:

wherein R₅—OH contains two hydroxyl groups (e.g., a diol, HO—R₅—OH),each of which are functionalized by reaction with an imide associatedwith R848. In some cases, HO—R₅—OH is a poly-diol such aspoly(hexamethyl carbonate) diol or polycaprolactone diol. For example,the reaction may be carried out as follows:

wherein the R groups are as described herein. Non-limiting examples ofsuitable polymers include polyketaldiols, poly(ethylene)glycol,polycaprolactone diol, diblock polylactide-co-poly(ethylene)glycol,diblock polylactide/polyglycolide-co-poly(ethylene)glycol, diblockpolyglycolide-co-poly(ethylene)glycol, poly(propylene) glycol,poly(hexamethylene carbonate)diol, and poly(tetrahydrofuran).

In embodiments where a poly-diol is employed, one of the diol groups maybe protected with a protecting group (e.g., t-butyloxycarbonyl), thusthe poly-diol would be a compound of formula HO—R₅—OP, wherein P is aprotecting group. Following reaction with an immunomodulatory agent toform a immunomodulatory agent-R₅—OP conjugate, the protecting group maybe removed and the second diol group may be reacted with any suitablereagent (e.g., PLGA, PLA).

Method 4

A conjugate (e.g., R848-PLA) can be formed via a one-pot ring-openingpolymerization of an immunomodulatory agent (e.g., R848) with a polymeror unit thereof (e.g., D/L-lactide) in the presence of a catalyst, forexample, as shown in the following scheme:

In a one-step procedure, the immunomodulatory agent and the polymer orunit thereof may be combined into a single reaction mixture comprising acatalyst. The reaction may proceed at a suitable temperature (e.g., atabout 150° C.) and the resulting conjugate may be isolated usingcommonly known techniques. Non-limiting examples of suitable catalystsinclude DMAP and tin ethylhexanoate.

Method 5

A conjugate can be formed via two-step ring opening polymerization of animmunomodulatory agent (e.g., R848) with one or more polymers or unitsthereof (e.g., D/L-lactide and glycolide) in the presence of a catalyst,for example, as shown in the following scheme:

The polymers or units thereof may be first combined, and in some cases,heated (e.g., to 135° C.) to form a solution. The immunomodulatory agentmay be added to a solution comprising the polymers or units thereof,followed by addition of a catalyst (e.g., tin ethylhexanoate). Theresulting conjugate may be isolated using commonly known techniques.Non-limiting examples of suitable catalysts include DMAP and tinethylhexanoate.

In some embodiments, a compound or conjugate provided herein, anotherimmunomodulatory agent, antigen, and/or targeting moiety can becovalently associated with a polymeric matrix. In some embodiments,covalent association is mediated by a linker. In some embodiments, acompound or conjugate provided herein, another immunomodulatory agent,antigen, and/or targeting moiety can be noncovalently associated with apolymeric matrix. For example, in some embodiments, a compound orconjugate provided herein, another immunomodulatory agent, antigen,and/or targeting moiety can be encapsulated within, surrounded by,and/or dispersed throughout a polymeric matrix. Alternatively oradditionally, a compound or conjugate provided herein, anotherimmunomodulatory agent, antigen, and/or targeting moiety can beassociated with a polymeric matrix by hydrophobic interactions, chargeinteractions, van der Waals forces, etc.

A wide variety of polymers and methods for forming polymeric matricestherefrom are known conventially. In general, a polymeric matrixcomprises one or more polymers. Polymers may be natural or unnatural(synthetic) polymers. Polymers may be homopolymers or copolymerscomprising two or more monomers. In terms of sequence, copolymers may berandom, block, or comprise a combination of random and block sequences.Typically, polymers in accordance with the present invention are organicpolymers.

Examples of polymers suitable for use in the present invention include,but are not limited to polyethylenes, polycarbonates (e.g.,poly(1,3-dioxan-2one)), polyanhydrides (e.g., poly(sebacic anhydride)),polyhydroxyacids (e.g., poly(β-hydroxyalkanoate)), polypropylfumerates,polycaprolactones, polyamides (e.g., polycaprolactam), polyacetals,polyethers, polyesters (e.g., polylactide, polyglycolide),poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polyureas, polystyrenes, polyamines, and polysaccharides (e.g.,chitosan).

In some embodiments, polymers in accordance with the present inventioninclude polymers which have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. §177.2600, includingbut not limited to polyesters (e.g., polylactic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymersmay comprise anionic groups (e.g., phosphate group, sulphate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group). In someembodiments, a synthetic nanocarrier comprising a hydrophilic polymericmatrix generates a hydrophilic environment within the syntheticnanocarrier. In some embodiments, polymers can be hydrophobic. In someembodiments, a synthetic nanocarrier comprising a hydrophobic polymericmatrix generates a hydrophobic environment within the syntheticnanocarrier. Selection of the hydrophilicity or hydrophobicity of thepolymer may have an impact on the nature of materials that areincorporated (e.g., coupled) within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. A variety of moieties or functional groups canbe used in accordance with the present invention. In some embodiments,polymers may be modified with PEG, with a carbohydrate, and/or withacyclic polyacetals derived from polysaccharides (Papisov, 2001, ACSSymposium Series, 786:301).

In some embodiments, polymers may be modified with a lipid or fatty acidgroup. In some embodiments, a fatty acid group may be one or more ofbutyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, polyanhydrides, poly(ortho ester),poly(ortho ester)-PEG copolymers, poly(caprolactone),poly(caprolactone)-PEG copolymers, polylysine, polylysine-PEGcopolymers, poly(ethyleneimine), poly(ethylene imine)-PEG copolymers,poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid],and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer may comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g., DNA, RNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 6:7), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc.Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897;Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,Bioconjugate Chem., 4:372) are positively-charged at physiological pH,form ion pairs with nucleic acids, and mediate transfection in a varietyof cell lines.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989,Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J.Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam etal., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem.Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al.,1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,121:5633).

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al.,2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc.,123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J.Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181).More generally, a variety of methods for synthesizing certain suitablepolymers are described in Concise Encyclopedia of Polymer Science andPolymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press,1980; Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step. It is further to beunderstood that inventive compounds and synthetic nanocarriers maycomprise block copolymers, graft copolymers, blends, mixtures, and/oradducts of any of the foregoing and other polymers. Those skilled in theart will recognize that the polymers listed herein represent anexemplary, not comprehensive, list of polymers that can be of use inaccordance with the present invention.

In some embodiments, synthetic nanocarriers may comprise metalparticles, quantum dots, ceramic particles, etc.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more amphiphilic entities. In some embodiments, an amphiphilic entitycan promote the production of synthetic nanocarriers with increasedstability, improved uniformity, or increased viscosity. In someembodiments, amphiphilic entities can be associated with the interiorsurface of a lipid membrane (e.g., lipid bilayer, lipid monolayer,etc.). Many amphiphilic entities known in the art are suitable for usein making synthetic nanocarriers in accordance with the presentinvention. Such amphiphilic entities include, but are not limited to,phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine(DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides;sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate(Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60);polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85(Tween®85); polyoxyethylene monostearate; surfactin; a poloxomer; asorbitan fatty acid ester such as sorbitan trioleate; lecithin;lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. An amphiphilic entity component may be a mixture of differentamphiphilic entities. Those skilled in the art will recognize that thisis an exemplary, not comprehensive, list of substances with surfactantactivity. Any amphiphilic entity may be used in the production ofsynthetic nanocarriers to be used in accordance with the presentinvention.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more carbohydrates. Carbohydrates may be natural or synthetic. Acarbohydrate may be a derivatized natural carbohydrate. In certainembodiments, a carbohydrate comprises monosaccharide or disaccharide,including but not limited to glucose, fructose, galactose, ribose,lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose,arabinose, glucoronic acid, galactoronic acid, mannuronic acid,glucosamine, galatosamine, and neuramic acid. In certain embodiments, acarbohydrate is a polysaccharide, including but not limited to pullulan,cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose(HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran,cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan, glycon,amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,starch, chitin, heparin, konjac, glucommannan, pustulan, heparin,hyaluronic acid, curdlan, and xanthan. In certain embodiments, thecarbohydrate is a sugar alcohol, including but not limited to mannitol,sorbitol, xylitol, erythritol, maltitol, and lactitol.

Synthetic nanocarriers may be prepared using a wide variety of methodsknown in the art. For example, synthetic nanocarriers can be formed bymethods as nanoprecipitation, flow focusing using fluidic channels,spray drying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanomaterials have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843). Additional methods havebeen described in the literature (see, e.g., Doubrow, Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press,Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13;Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz etal., 1988, J. Appl. Polymer Sci., 35:755, and also U.S. Pat. Nos.5,578,325 and 6,007,845).

In certain embodiments, synthetic nanocarriers are prepared by ananoprecipitation process or spray drying. Conditions used in preparingsynthetic nanocarriers may be altered to yield particles of a desiredsize or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness,” shape, etc.). The method of preparing thesynthetic nanocarriers and the conditions (e.g., solvent, temperature,concentration, air flow rate, etc.) used may depend on the materials tobe coupled to the synthetic nanocarriers and/or the composition of thepolymer matrix.

If particles prepared by any of the above methods have a size rangeoutside of the desired range, particles can be sized, for example, usinga sieve.

Coupling can be achieved in a variety of different ways, and can becovalent or non-covalent. Such couplings may be arranged to be on asurface or within an inventive synthetic nanocarrier. Elements of theinventive synthetic nanocarriers (such as moieties of which animmunofeature surface is comprised, targeting moieties, polymericmatrices, and the like) may be directly coupled with one another, e.g.,by one or more covalent bonds, or may be coupled by means of one or morelinkers. Additional methods of functionalizing synthetic nanocarriersmay be adapted from Published US Patent Application 2006/0002852 toSaltzman et al., Published US Patent Application 2009/0028910 toDeSimone et al., or Published International Patent ApplicationWO/2008/127532 A1 to Murthy et al.

Any suitable linker can be used in accordance with the presentinvention. Linkers may be used to form amide linkages, ester linkages,disulfide linkages, etc. Linkers may contain carbon atoms or heteroatoms(e.g., nitrogen, oxygen, sulfur, etc.). In some embodiments, a linker isan aliphatic or heteroaliphatic linker. In some embodiments, the linkeris a polyalkyl linker. In certain embodiments, the linker is a polyetherlinker. In certain embodiments, the linker is a polyethylene linker. Incertain specific embodiments, the linker is a polyethylene glycol (PEG)linker.

In some embodiments, the linker is a cleavable linker. To give but a fewexamples, cleavable linkers include protease cleavable peptide linkers,nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers,glycosidase sensitive carbohydrate linkers, pH sensitive linkers,hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers,enzyme cleavable linkers (e.g., esterase cleavable linker),ultrasound-sensitive linkers, x-ray cleavable linkers, etc. In someembodiments, the linker is not a cleavable linker.

A variety of methods can be used to couple a linker or other element ofa synthetic nanocarrier with the synthetic nanocarrier. Generalstrategies include passive adsorption (e.g., via electrostaticinteractions), multivalent chelation, high affinity non-covalent bindingbetween members of a specific binding pair, covalent bond formation,etc. (Gao et al., 2005, Curr. Op. Biotechnol., 16:63). In someembodiments, click chemistry can be used to associate a material with asynthetic nanocarrier.

Non-covalent specific binding interactions can be employed. For example,either a particle or a biomolecule can be functionalized with biotinwith the other being functionalized with streptavidin. These twomoieties specifically bind to each other noncovalently and with a highaffinity, thereby associating the particle and the biomolecule. Otherspecific binding pairs could be similarly used. Alternately,histidine-tagged biomolecules can be associated with particlesconjugated to nickel-nitrolotriaceteic acid (Ni-NTA).

For additional general information on coupling, see the journalBioconjugate Chemistry, published by the American Chemical Society,Columbus Ohio, PO Box 3337, Columbus, Ohio, 43210; “Cross-Linking,”Pierce Chemical Technical Library, available at the Pierce web site andoriginally published in the 1994-95 Pierce Catalog, and references citedtherein; Wong S S, Chemistry of Protein Conjugation and Cross-linking,CRC Press Publishers, Boca Raton, 1991; and Hermanson, G. T.,Bioconjugate Techniques, Academic Press, Inc., San Diego, 1996.

It is to be understood that the compositions of the invention can bemade in any suitable manner, and the invention is in no way limited tocompositions that can be produced using the methods described herein.Selection of an appropriate method may require attention to theproperties of the particular moieties being associated.

Pharmaceutical Compositions and Methods of Use

Compositions according to the invention comprise inventive compounds,conjugates, or synthetic nanocarriers, optionally, in combination withpharmaceutically acceptable excipients. The compositions may be madeusing conventional pharmaceutical manufacturing and compoundingtechniques to arrive at useful dosage forms. In an embodiment, inventivecompounds, conjugates, synthetic nanocarriers, or compositions aresuspended in sterile saline solution for injection together with apreservative.

In some embodiments, inventive compounds, conjugates, syntheticnanocarriers, or compositions are manufactured under sterile conditionsor are terminally sterilized. This can ensure that resultingcompositions are sterile and non-infectious, thus improving safety whencompared to non-sterile compositions. This provides a valuable safetymeasure, especially when subjects receiving inventive compounds,conjugates, synthetic nanocarriers, or compositions have immune defects,are suffering from infection, and/or are susceptible to infection. Insome embodiments, inventive compounds, conjugates, syntheticnanocarriers, or compositions may be lyophilized and stored insuspension or as lyophilized powder depending on the formulationstrategy for extended periods without losing activity.

The inventive compounds, conjugates, synthetic nanocarriers, orcompositions may be administered by a variety of routes ofadministration, including but not limited to parenteral, subcutaneous,intramuscular, intradermal, oral, intranasal, transmucosal, rectal;ophthalmic, transdermal, transcutaneous or by a combination of theseroutes.

The inventive compounds, conjugates, synthetic nanocarriers, orcompositions and methods described herein can be used to induce,enhance, stimulate, modulate, or direct an immune response. Theinventive compounds, conjugates, synthetic nanocarriers, or compositionsand methods described herein can be used in the diagnosis, prophylaxisand/or treatment of conditions such as cancers, infectious diseases,metabolic diseases, degenerative diseases, inflammatory diseases,immunological diseases, or other disorders and/or conditions. Theinventive compounds, conjugates, synthetic nanocarriers, or compositionsand methods described herein can also be used for the prophylaxis ortreatment of an addiction, such as an addiction to nicotine or anarcotic. The inventive compounds, conjugates, synthetic nanocarriers,or compositions and methods described herein can also be used for theprophylaxis and/or treatment of a condition resulting from the exposureto a toxin, hazardous substance, environmental toxin, or other harmfulagent.

EXAMPLES Example 1: One-Pot Ring-Opening Polymerization of R848 withD/L-Lactide in the Presence of a Catalyst

A mixture of R848 (0.2 mmol, 63 mg), D/L-lactide (40 mmol, 5.8 g), and4-dimethylaminopyridine (DMAP) (50 mg, 0.4 mmol) in 2 mL of anhydroustoluene was heated slowly to 150° C. (oil bath temperature) andmaintained at this temperature for 18 h (after 3 hr, no R848 was left).The mixture was cooled to ambient temperature and the resulting mixturewas quenched with water (50 mL) to precipitate out the resultingpolymer, R848-PLA. The polymer was then washed sequentially with 45 mLeach of MeOH, iPrOH, and ethyl ether. The polymer was dried under vacuumat 30° C. to give an off-white puffy solid (5.0 g). Polymeric structurewas confirmed by ¹H NMR in CDCl₃. A small sample of the polymer wastreated with 2 N NaOH aq in THF/MeOH to determine the loading of R848 onthe polymer by reverse phase HPLC. The loading of R848 is 3 mg per gramof polymer (0.3% loading-27.5% of theory).

Example 2: Two Step Ring Opening Polymerization of R848 with D/L-Lactideand Glycolide

A mixture of D/L-lactide (10.8 g, 0.075 moles) and glycolide (2.9 g,0.025 moles) was heated to 135° C. under argon. Once all of thematerials had melted and a clear solution had resulted, R848 (1.08 g,3.43×10⁻³ moles) was added. This solution was stirred at 135° C. under aslow stream of argon for one hour. Tin ethylhexanoate (150 μL) was addedand heating was continued for 4 hours. After cooling, the solid palebrown mass was dissolved in methylene chloride (250 mL) and the solutionwas washed with 5% tartaric acid solution (2×200 mL). The methylenechloride solution was dried over magnesium sulfate, filtered, and thenconcentrated under vacuum. The residue was dissolved in methylenechloride (20 mL) and 2-propanol (250 mL) was added with stirring. Thepolymer that separated was isolated by decantation of the 2-propanol andwas dried under high vacuum. NMR showed that the polymer was 71.4%lactide and 28.6% glycolide with a molecular weight of 4000. The loadingof R848 was close to theoretical by NMR.

Example 3: Preparation of PLGA-R848 Conjugate

A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number0.7 mmol/g, 10 g, 7.0 mmol) and HBTU (5.3 g, 14 mmol) in anhydrous EtOAc(160 mL) was stirred at room temperature under argon for 50 minutes.Compound R848 (2.2 g, 7 mmol) was added, followed bydiisopropylethylamine (DIPEA) (5 mL, 28 mmol). The mixture was stirredat room temperature for 6 h and then at 50-55° C. overnight (about 16h). After cooling, the mixture was diluted with EtOAc (200 mL) andwashed with saturated NH₄Cl solution (2×40 mL), water (40 mL) and brinesolution (40 mL). The solution was dried over Na₂SO₄ (20 g) andconcentrated to a gel-like residue. Isopropyl alcohol (IPA) (300 mL) wasthen added and the polymer conjugate precipitated out of solution. Thepolymer was then washed with IPA (4×50 mL) to remove residual reagentsand dried under vacuum at 35-40° C. for 3 days as a white powder (10.26g, MW by GPC is 5200, R848 loading is 12% by HPLC).

Example 4: Preparation of PLGA-854a Conjugate

A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrousEtOAc (20 mL) was stirred at room temperature under argon for 45minutes. Compound 845A (0.29 g, 0.7 mmol) was added, followed bydiisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). The mixture wasstirred at room temperature for 6 h and then at 50-55° C. overnight(about 15 h). After cooling, the mixture was diluted with EtOAc (100 mL)and washed with saturated NH4Cl solution (2×20 mL), water (20 mL) andbrine solution (20 mL). The solution was dried over Na₂SO₄ (10 g) andconcentrated to a gel-like residue. Isopropyl alcohol (IPA) (40 mL) wasthen added and the polymer conjugate precipitated out of solution. Thepolymer was then washed with IPA (4×25 mL) to remove residual reagentsand dried under vacuum at 35-40° C. for 2 days as a white powder (1.21g, MW by GPC is 4900, 854A loading is 14% by HPLC).

Example 5: Preparation of PLGA-BBHA Conjugate

A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrousEtOAc (30 mL) was stirred at room temperature under argon for 30minutes. Compound BBHA (0.22 g, 0.7 mmol) in 2 mL of dry DMSO was added,followed by diisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). Themixture was stirred at room temperature for 20 h. Additional amounts ofHBTU (0.53 g, 1.4 mmol) and DIPEA (0.5 mL, 2.8 mmol) were added and themixture was heated at 50-55° C. for 4 h. After cooling, the mixture wasdiluted with EtOAc (100 mL) and washed with saturated NH4Cl solution 20mL), water (2×20 mL) and brine solution (20 mL). The solution was driedover Na₂SO₄ (10 g) and concentrated to a gel-like residue. Isopropylalcohol (IPA) (35 mL) was then added and the brownish polymer conjugateprecipitated out of solution. The polymer was then washed with IPA (2×20mL) to remove residual reagents and dried under vacuum at 35-40° C. for2 days as a brownish powder (1.1 g).

Example 6: Preparation of Low MW PLA-R848 Conjugate

A solution of PLA-CO2H (average MW: 950, DPI: 1.32; 5.0 g, 5.26 mmol)and HBTU (4.0 g, 10.5 mmol) in EtOAc (120 mL) was stirred at roomtemperature under argon for 45 min. Compound R848 (1.65 g, 5.26 mmol)was added, followed by DIPEA (5.5 mL, 31.6 mmol). The mixture wasstirred at room temperature for 6 h and then at 50-55° C. for 15 h.After cooling, the mixture was diluted with EtOAc (150 mL) and washedwith 1% citric acid solution (2×40 mL), water (40 mL) and brine solution(40 mL). The solution was dried over Na₂SO₄ (10 g) and concentrated to agel-like residue. Methyl t-butyl ether (MTBE) (150 mL) was then addedand the polymer conjugate precipitated out of solution. The polymer wasthen washed with MTBE (50 mL) and dried under vacuum at room temperaturefor 2 days as a white foam (5.3 g, average MW by GPC is 1200, PDI: 1.29;R848 loading is 20% by HPLC).

Example 7: Preparation of Low MW PLA-R848 Conjugate

A solution of PLA-CO2H (average MW: 1800, DPI: 1.44; 9.5 g, 5.26 mmol)and HBTU (4.0 g, 10.5 mmol) in EtOAc (120 mL) was stirred at roomtemperature under argon for 45 min. Compound R848 (1.65 g, 5.26 mmol)was added, followed by DIPEA (5.5 mL, 31.6 mmol). The mixture wasstirred at room temperature for 6 h and then at 50-55° C. for 15 h.After cooling, the mixture was diluted with EtOAc (150 mL) and washedwith 1% citric acid solution (2×40 mL), water (40 mL) and brine solution(40 mL). The solution was dried over Na₂SO₄ (10 g) and concentrated to agel-like residue. Methyl t-butyl ether (MTBE) (150 mL) was then addedand the polymer conjugate precipitated out of solution. The polymer wasthen washed with MTBE (50 mL) and dried under vacuum at room temperaturefor 2 days as a white foam (9.5 g, average MW by GPC is 1900, PDI: 1.53;R848 loading is 17% by HPLC).

Example 8: Conjugation of R848 to PCADK Via Imide Ring Opening

The following examples describes the synthesis of a polyketal, PCADK,according to a method provided in Pulendran et al, WO 2008/127532,illustrated in step 1 below.

PCADK is synthesized in a 50 mL two-necked flask, connected to ashort-path distilling head. First, 5.5 mg of re-crystallizedp-toluenesulfonic acid (0.029 mmol, Aldrich, St. Louis, Mo.), isdissolved in 6.82 mL of ethyl acetate, and added to a 30 mL benzenesolution (kept at 100° C.), which contains 1,4-cyclohexanedimethanol(12.98 g, 90.0 mmol, Aldrich). The ethyl acetate is allowed to boil off,and distilled 2,2-dimethoxypropane (10.94 mL, 90.0 mmol, Aldrich) isadded to the benzene solution, initiating the polymerization reaction.Additional doses of 2,2-dimethoxypropane (5 mL) and benzene (25 mL) aresubsequently added to the reaction every hour for 6 hours via a meteringfunnel to compensate for 2,2-dimethoxypropane and benzene that isdistilled off. After 8 hours, the reaction is stopped by addition of 500μL of triethylamine. The polymer is isolated by precipitation in coldhexane (stored at −20° C.) followed by vacuum filtration. The molecularweight of PCADK is determined by gel permeation chromatography (GPC)(Shimadzu, Kyoto, Japan) equipped with a UV detector. THF is used as themobile phase at a flow rate of 1 ml/min. Polystyrene standards fromPolymer Laboratories (Amherst, Mass.) are used to establish a molecularweight calibration curve. This compound is used to generate the PCADKparticles in all subsequent experiments.

R848 may be conjugated to the terminal alcohol groups of the PCADKhaving molecular weight 6000 via imide ring opening, according to thestep 2 shown below.

Step 1: Preparation of PCADK

Step 2: Conjugation of PCADK to R848

In step 2, the polymer from step 1 (12 g, 2.0×10⁻³ moles) is dissolvedin methylene chloride 100 mL, and the lactam of R848 (3.3 g, 8.0×10⁻³moles) is added. This slurry is stirred as1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.835 g, 6×10⁻³ moles) isadded in a single portion. After stirring at room temperature overnight,a clear solution forms. The solution is diluted with methylene chloride(100 mL) and the solution is washed with 5% citric acid. This solutionis dried over sodium sulfate after which it is filtered and evaporatedunder vacuum. After drying under high vacuum there is obtained 11.3grams (81%) of polymer. A portion is hydrolyzed in acid and the R848content is determined to be 9% by weight.

Example 9: Conjugation of R848 to Poly-Caprolactonediol Via Imide RingOpening

Imide ring opening is used to attach R854 to the terminal alcohol groupsof poly-caprolactonediol of molecular weight 2000. The polycaprolactonediol is purchased from Aldrich Chemical Company, Cat. #189421, and hasthe following structure:

The polycaprolactone diol-R854 conjugate has the following structure:

The polymer (5 g, 2.5×10⁻³ moles) is dissolved in methylene chloride 25mL and the lactam of R854 (2.4 g, 5.0×10⁻³ moles) is added. This slurryis stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4×10⁻³moles) is added in a single portion. After stirring at room temperaturefor 15 minutes, a clear pale yellow solution forms. The solution isdiluted with methylene chloride (100 mL) and the solution is washed with5% citric acid. This solution is dried over sodium sulfate after whichit is filtered and evaporated under vacuum. After drying under highvacuum there is obtained 5.2 grams (70%) of polymer. A portion ishydrolyzed in acid and the R848 content is determined to be 18.5% byweight.

Example 10: Conjugation of R848 to Poly-(Hexamethylene Carbonate)DiolVia Imide Ring Opening

Imide ring opening is used to attach R848 to the terminal alcohol groupsof poly-(hexamethylene carbonate)diol of molecular weight 2000. Thepoly(hexamethylene carbonate) diol is purchased from Aldrich ChemicalCompany, Cat #461164, and has the following structure:HO—[CH₂(CH₂)₄CH₂OCO₂ ]nCH₂(CH₂)₄CH₂—OH.

The poly(hexamethylene carbonate) diol-R848 conjugate has the followingstructure:

The polymer (5 g, 2.5×10⁻³ moles) is dissolved in methylene chloride 25mL and the lactam of R848 (2.06 g, 5.0×10⁻³ moles) is added. This slurryis stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4×10⁻³moles) is added in a single portion. After stirring at room temperatureovernight a clear pale yellow solution forms. The solution is dilutedwith methylene chloride (100 mL) and the solution is washed with 5%citric acid. This solution is dried over sodium sulfate after which itis filtered and evaporated under vacuum. After drying under high vacuumthere is obtained 5.9 grams (84%) of polymer. NMR is used to determinethe R848 content which is determined to be 21%.

Example 11: Polylactic Acid Conjugates of an Imidazoquinoline Using aTin Ethylhexanoate Catalyst

To a two necked round bottom flask equipped with a stir bar andcondenser was added the imidazoquinoline resiquimod (R-848, 100 mg,3.18×10⁻⁴ moles), D/L lactide (5.6 g, 3.89×10⁻² moles) and anhydroussodium sulfate (4.0 g). The flask and contents were dried under vacuumat 50° C. for 8 hours. The flask was then flushed with argon and toluene(100 mL) was added. The reaction was stirred in an oil bath set at 120°C. until all of the lactide had dissolved and then tin ethylhexanoate(75 mg, 60 μL) was added via pipette. Heating was continued under argonfor 16 hours. After cooling, water (20 mL) was added and stirring wascontinued for 30 minutes. The reaction was diluted with additionaltoluene (200 mL) and was then washed with water (200 mL). The toluenesolution was then washed in turn with 10% sodium chloride solutioncontaining 5% conc. Hydrochloric acid (200 mL) followed by saturatedsodium bicarbonate (200 mL). TLC (silica, 10% methanol in methylenechloride) showed that the solution contained no free R-848. The solutionwas dried over magnesium sulfate, filtered and evaporated under vacuumto give 3.59 grams of polylactic acid-R-848 conjugate. A portion of thepolymer was hydrolyzed in base and examined by HPLC for R-848 content.By comparison to a standard curve of R-848 concentration vs. HPLCresponse, it was determined that the polymer contained 4.51 mg of R-848per gram of polymer. The molecular weight of the polymer was determinedby GPC to be about 19,000.

Example 12: Low Molecular Weight Polylactic Acid Conjugates of anImidazoquinoline

To a round bottom flask equipped with a stir bar and condenser was addedthe imidazoquinoline, resiquimod (R-848, 218 mg, 6.93×10⁻⁴ moles), D/Llactide (1.0 g, 6.93×10⁻³ moles) and anhydrous sodium sulfate (800 mg).The flask and contents were dried under vacuum at 55° C. for 8 hours.After cooling, the flask was then flushed with argon and toluene (50 mL)was added. The reaction was stirred in an oil bath set at 120° C. untilall of the lactide had dissolved and then tin ethylhexanoate (19 mg, 15μL) was added via pipette. Heating was continued under argon for 16hours. After cooling, the reaction was diluted with ether (200 mL) andthe solution was washed with water (200 mL). The solution was dried overmagnesium sulfate, filtered and evaporated under vacuum to give 880 mg.of crude polylactic acid-R-848 conjugate. The crude polymer waschromatographed on silica using 10% methanol in methylene chloride aseluent. The fractions containing the conjugate were pooled andevaporated to give the purified conjugate. This was dried under highvacuum to provide the conjugate as a solid foam in a yield of 702 mg(57.6%). By integrating the NMR signals for the aromatic protons of thequinoline and comparing this to the integrated intensity of the lacticacid CH proton it was determined that the molecular weight of theconjugate was approximately 2 KD. GPC showed that the conjugatecontained less than 5% of free R848.

Example 13: Low Molecular Weight Polylactic Acid Co-Glycolic AcidConjugates of an Imidazoquinoline

To a round bottom flask equipped with a stir bar and condenser was addedthe imidazoquinoline, resiquimod (R-848, 436 mg, 1.39×10⁻³ moles),glycolide (402 mg, 3.46×10⁻³ moles), D/L lactide (2.0 g, 1.39×10⁻²moles) and anhydrous sodium sulfate (1.6 g). The flask and contents weredried under vacuum at 55° C. for 8 hours. After cooling, the flask wasthen flushed with argon and toluene (60 mL) was added. The reaction wasstirred in an oil bath set at 120° C. until all of the R848, glycolideand lactide had dissolved and then tin ethylhexanoate (50 mg, 39 μL) wasadded via pipette. Heating was continued under argon for 16 hours. Aftercooling, the reaction was diluted with ethyl acetate (200 mL) and thesolution was washed with water (200 mL). The solution was dried overmagnesium sulfate, filtered and evaporated under vacuum to give crudePLGA-R-848 conjugate. The crude polymer was chromatographed on silicausing 10% methanol in methylene chloride as eluent. The fractionscontaining the conjugate were pooled and evaporated to give the purifiedconjugate. This was dried under high vacuum to provide the conjugate asa solid foam in a yield of 1.55 g (54.6%). By integrating the NMRsignals for the aromatic protons of the quinoline and comparing this tothe integrated intensity of the lactic acid CH proton it was determinedthat the molecular weight of the conjugate was approximately 2 KD. GPCshowed that the conjugate contained no detectable free R848.

Example 14: Polylactic Acid Conjugates of an Imidazoquinoline Using aLithium Diisopropylamide Catalysis

The imidazoquinoline (R-848), D/L lactide, and associated glassware wereall dried under vacuum at 50° C. for 8 hours prior to use. To a roundbottom flask equipped with a stir bar and condenser was added the R-848(33 mg, 1.05×10⁻⁴ moles), and dry toluene (5 mL). This was heated toreflux to dissolve all of the R-848. The solution was stirred undernitrogen and cooled to room temperature to provide a suspension offinely divided R-848. To this suspension was added a solution of lithiumdiisopropyl amide (2.0 M in THF, 504, 1.0×10⁻⁴ moles) after whichstirring was continued at room temperature for 5 minutes. The paleyellow solution that had formed was added via syringe to a hot (120° C.)solution of D/L lactide (1.87 g, 1.3×10⁻² moles) under nitrogen. Theheat was removed and the pale yellow solution was stirred at roomtemperature for one hour. The solution was diluted with methylenechloride (200 mL) and this was then washed with 1% hydrochloric acid(2×50 mL) followed by saturated sodium bicarbonate solution (50 mL). Thesolution was dried over magnesium sulfate, filtered and evaporated undervacuum to give the polylactic acid-R-848 conjugate. TLC (silica, 10%methanol in methylene chloride) showed that the solution contained nofree R-848. The polymer was dissolved in methylene chloride (10 mL) andthe solution was dripped into stirred hexane (200 mL). The precipitatedpolymer was isolated by decantation and was dried under vacuum to give1.47 grams of the polylactic acid—R-848 conjugate as a white solid. Aportion of the polymer was hydrolyzed in base and examined by HPLC forR-848 content. By comparison to a standard curve of R-848 concentrationvs. HPLC response, it was determined that the polymer contained 10.96 mgof R-848 per gram of polymer.

Example 15: Polylactic Acid Activation

PLA (D/L-polylactide) (Resomer R202H from Boehringer-Ingelheim, KOHequivalent acid number of 0.21 mmol/g, intrinsic viscosity (iv): 0.21dl/g) (10 g, 2.1 mmol, 1.0 eq) was dissolved in dichloromethane (DCM)(35 mL). EDC (2.0 g, 10.5 mmol, 5 eq) and NHS (1.2 g, 10.5 mmol, 5 eq)were added. The solids were dissolved with the aid of sonication. Theresulting solution was stirred at room temperature for 6 days. Thesolution was concentrated to remove most of DCM and the residue wasadded to a solution of 250 mL of diethyl ether and 5 mL of MeOH toprecipitate out the activated PLA-NHS ester. The solvents were removedand the polymer was washed twice with ether (2×200 mL) and dried undervacuum to give PLA-NHS activated ester as a white foamy solid (˜8 grecovered, ¹H NMR confirmed the presence of NHS ester). The PLA-NHSester is stored under argon in a below −10° C. freezer before use.

Alternatively, the reaction can be performed in DMF, THF, dioxane, orCHCl₃ instead of DCM. DCC can be used instead of EDC (resulting DCC-ureais filtered off before precipitation of the PLA-NHS ester from ether).The amount of EDC or DCC and NHS can be in the range of 2-10 eq of thePLA.

Example 16: PLA Activation

PLA (D/L-polylactide) with MW of 5000 (10.5 g, 2.1 mmol, 1.0 eq) isdissolved in dichloromethane (DCM) (35 mL). EDC (2.0 g, 10.5 mmol, 5 eq)and NHS (1.2 g, 10.5 mmol, 5 eq) are added. The resulting solution isstirred at room temperature for 3 days. The solution is concentrated toremove most of DCM and the residue is added to a solution of 250 mL ofdiethyl ether and 5 mL of MeOH to precipitate out the activated PLA-NHSester. The solvents are removed and the polymer is washed twice withether (2×200 mL) and dried under vacuum to give PLA-NHS activated esteras a white foamy solid (˜8 g recovered, H NMR can be used to confirm thepresence of NHS ester). The PLA-NHS ester is stored under argon in abelow −10° C. freezer before use.

Alternatively, the reaction can be performed in DMF, THF, dioxane, orCHCl3 instead of DCM. DCC can be used instead of EDC (resulting DCC-ureais filtered off before precipitation of the PLA-NHS ester from ether).The amount of EDC or DCC and NHS can be in the range of 2-10 eq of thePLA.

Example 17: Low MW PLGA Activation

In the same manner as provided above for polymer activation, low MW PLGAwith 50% to 75% glycolide is converted to the corresponding PLGA-NHSactivated ester and is stored under argon in a below −10° C. freezerbefore use.

Example 18: Polylactic Acid Activation

PLA (R202H, acid number of 0.21 mmol/g) (2.0 g, 0.42 mmol, 1.0 eq) wasdissolved in 10 mL of dry acetonitrile. N,N′-disuccinimidyl carbonate(DSC) (215 mg, 1.26 mmol, 3.0 eq) and catalytic amount of4-(N,N-dimethylamino)pyridine (DMAP) were added. The resulting mixturewas stirred under argon for 1 day. The resulting solution wasconcentrated to almost dryness. The residue was then added to 40 mL ofether to precipitate out the polymer which was washed twice with ether(2×30 mL) and dried under vacuum to give PLA-NHS activated ester (1H NMRshowed the amount of NHS ester at about 80%).

Example 19: Polylactic Acid Activation

PLA (R202H) (5.0 g, 1.05 mmol) was dissolved in 25 mL of anhydrous DCMand 2.5 mL of anhydrous DMF. DCC (650 mg, 3.15 mmol, 5.0 eq) andpentafluorophenol (PFP) (580 mg, 3.15 mmol, 5.0 eq) were added. Theresulting solution was stirred at room temperature for 6 days and thenconcentrated to remove DCM. The resulting residue was added to 250 mL ofether to precipitate out the activated PLA polymer which was washed withether (2×100 mL) and dried under vacuum to give PLA-PFP activated esteras a white foamy solid (4.0 g).

Example 20: Polylactic Acid or PLGA Conjugates of an Imidazoquinoline

PLA-NHS (1.0 g), R848 (132 mg, 0.42 mmol), and diisopropylethylamine(DIPEA) (0.073 mL, 0.42 mmol) were dissolved in 2 mL of dry DMF underargon. The resulting solution was heated at 50-60° C. for 2 days. Thesolution was cooled to room temperature and added to 40 mL of de-ionized(DI) water to precipitate out the polymer product. The polymer was thenwashed with DI water (40 mL) and ether (2×40 mL) and dried at 30° C.under vacuum to give R848-PLA conjugate as a white foamy solid (0.8 g, HNMR showed the conjugation of R848 to PLA via the amide bond). Thedegree of conjugation (loading) of R848 on the polymer was confirmed byHPLC analysis as follows: a weighed amount of polymer was dissolved inTHF/MeOH and treated with 15% NaOH. The resulting hydrolyzed polymerproducts were analyzed for the amount of R848 by HPLC in comparison witha standard curve.

Example 21: Polylactic Acid or PLGA Conjugates of an Imidazoquinoline

PLA-NHS (1.0 g, 0.21 mmol, 1.0 eq), R848 (132 mg, 0.42 mmol, 2.0 eq),DIPEA (0.15 mL, 0.84 mmol, 4.0 eq) and DMAP (25 mg, 0.21 mmol, 1.0 eq)were dissolved in 2 mL of dry DMF under argon. The resulting solutionwas heated at 50-60° C. for 2 days. The solution was cooled to roomtemperature and added to 40 mL of de-ionized (DI) water to precipitateout the polymer product. The polymer was then washed with DI water (40mL) and ether (2×40 mL) and dried at 30° C. under vacuum to givePLA-R848 conjugate as a white foamy solid (0.7 g, 20 mg of the polymerwas hydrolyzed in solution of 0.2 mL of THF, 0.1 mL of MeOH and 0.1 mLof 15% NaOH. The amount of R848 on the polymer was determined to beabout 35 mg/g by reverse phase HPLC analysis (C18 column, mobile phaseA: 0.1% TFA in water, mobile phase B: 0.1% TFA in CH3CN, gradient).

Example 22: Polylactic Acid Conjugates of an Imidazoquinoline

PLA (R202H) (2.0 g, 0.42 mmol, 1.0 eq), DCC (260 mg, 1.26 mmol, 3.0 eq),NHS (145 mg, 1.26 mmol, 3.0 eq), R848 (200 mg, 0.63 mmol, 1.5 eq), DMAP(77 mg, 0.63 mmol, 1.5 eq) and DIPEA (0.223 mL, 1.26 mmol, 3.0 eq) weredissolved in 4 mL of dry DMF. The mixture was heated at 50-55° C. for 3days. The mixture was cooled to room temperature and diluted with DCM.The DCC-urea was filtered off and the filtrate was concentrated toremove DCM. The resulting residue in DMF was added to water (40 mL) toprecipitate out the polymer product which was washed with water (40 mL),ether/DCM (40 mL/4 mL) and ether (40 mL). After drying under vacuum at30° C., the desired PLA-R848 conjugate was obtained as a white foamysolid (1.5 g).

Example 23: Polylactic Acid Conjugates of an Imidazoquinoline

PLA (R202H) (2.0 g, 0.42 mmol, 1.0 eq), EDC (242 mg, 1.26 mmol, 3.0 eq),HOAt (171 mg, 1.26 mmol, 3.0 eq), R848 (200 mg, 0.63 mmol, 1.5 eq), andDIPEA (0.223 mL, 1.26 mmol, 3.0 eq) were dissolved in 4 mL of dry DMF.The mixture was heated at 50-55° C. for 2 days. The solution was cooledto room temperature and added to water (40 mL) to precipitate out thepolymer product which was washed with water (40 mL), ether/MeOH (40 mL/2mL) and ether (40 mL). The orange colored polymer was dissolved in 4 mLof DCM and the resulting solution was added to 40 mL of ether toprecipitate out the polymer without much of the orange color. The lightcolored polymer was washed with ether (40 mL). After drying under vacuumat 30° C., the desired PLA-R848 conjugate was obtained as a light brownfoamy solid (1.5 g).

Example 24: Polylactic Acid or PLGA Conjugates of an Imidazoquinoline

PLA (R202H) (1.0 g, 0.21 mmol, 1.0 eq), EDC (161 mg, 0.84 mmol, 4.0 eq),HOBt.H2O (65 mg, 0.42 mmol, 2.0 eq), R848 (132 mg, 0.42 mmol, 2.0 eq),and DIPEA (0.150 mL, 0.84 mmol, 4.0 eq) were dissolved in 2 mL of dryDMF. The mixture was heated at 50-55° C. for 2 days. The solution wascooled to room temperature and added to water (40 mL) to precipitate outthe polymer product. The orange colored polymer was dissolved in 2 mL ofDCM and the resulting solution was added to 40 mL of ether toprecipitate out the polymer which was washed with water/acetone (40 mL/2mL) and ether (40 mL). After drying under vacuum at 30° C., the desiredPLA-R848 conjugate was obtained as an off-white foamy solid (1.0 g,loading of R848 on polymer was about 45 mg/g based on HPLC analysis andconfirmed by ¹H NMR). In the same manner, PLGA (75% Lactide)-R848 andPLGA (50% lactide)-R848 were prepared.

Example 25: Conjugation of R848 to Polyglycine, a Polyamide

The t-butyloxycarbonyl (tBOC) protected polyglycine carboxylic acid (I)is prepared by ring opening polymerization of glycine N-carboxyanhydride(Aldrich cat #369772) using 6-aminohexanoic acid benzyl ester (Aldrichcat #S33465) by the method of Aliferis et al. (Biomacromolecules, 5,1653, (2004)). Protection of the end amino group as the t-BOC carbamatefollowed by hydrogenation over palladium on carbon to remove the benzylester completes the synthesis of BOC protected polyglycine carboxylicacid (I).

A mixture of BOC-protected polyglycine carboxylic acid (5 gm, MW=2000,2.5×10⁻³ moles) and HBTU (3.79 gm, 1.0×10⁻² moles) in anhydrous DMF (100mL) is stirred at room temperature under argon for 50 minutes. Then R848(1.6 gm, 5.0×10⁻³ moles) is added, followed by diisopropylethylamine (4mL, 2.2×10⁻² moles). The mixture is stirred at RT for 6 h and then at50-55° C. overnight (16 h). After cooling, the DMF is evaporated undervacuum and the residue is triturated in EtOAc (100 mL). The polymer isisolated by filtration and the polymer is then washed with 2-propanol(4×25 mL) to remove residual reagents and dried under vacuum at 35-40°C. for 3 days. The polymer is isolated as an off white solid in a yieldof 5.1 g (88%). The R848 loading that can be determined by NMR is 10.1%.

The t-BOC protecting group is removed using trifluoroacetic acid and theresulting polymer is grafted to PLA with carboxyl end groups byconventional methods.

Example 26: Preparation of a PLGA Conjugate of the Polyglycine/R848Polymer

Step 1: A t-BOC protected polyglycine/R848 conjugate (5 g) is dissolvedin trifluoroacetic acid (25 mL) and this solution is warmed at 50° C.for one hour. After cooling, the trifluoroacetic acid is removed undervacuum and the residue is triturated in ethyl acetate (25 mL). Thepolymer is isolated by filtration and is washed well with 2-propanol.After drying under vacuum there is obtained 4.5 grams of polymer as anoff white solid.

Step 2: A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A,acid number 0.7 mmol/g, 10 g, 7.0 mmol) and HBTU (5.3 g, 14 mmol) inanhydrous DMF (100 mL) is stirred at RT under argon for 50 minutes. Thepolymer from above (1.4 g, 7 mmol) dissolved in dry DMF (20 mL) isadded, followed by diisopropylethylamine (DIPEA) (5 mL, 28 mmol). Themixture is stirred at RT for 6 h and then at 50-55° C. overnight (16 h).After cooling, the DMF is evaporated under vacuum, and the residue isdissolved in methylene chloride (50 mL). The polymer is precipitated bythe addition of 2-propanol (200 mL). The polymer is isolated bydecantation and is washed with 2-propanol (4×50 mL) to remove residualreagents and then dried under vacuum at 35-40 C overnight. There isobtained 9.8 g (86%) of the block copolymer.

Example 27: Preparation of PLGA-2-Butoxy-8-Hydroxy-9-Benzyl AdenineConjugate

A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrousEtOAc (30 mL) is stirred at RT under argon for 30 minutes. Compound (I)(0.22 g, 0.7 mmol) in 2 mL of dry DMSO is added, followed bydiisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). The mixture isstirred at room temperature for 20 h. Additional amounts of HBTU (0.53g, 1.4 mmol) and DIPEA (0.5 mL, 2.8 mmol) are added and the mixture isheated at 50-55° C. for 4 h. After cooling, the mixture is diluted withEtOAc (100 mL) and washed with saturated NH₄Cl solution 20 mL), water(2×20 mL) and brine solution (20 mL). The solution is dried over Na₂SO₄(10 g) and concentrated to a gel-like residue. Isopropyl alcohol (IPA)(35 mL) is then added and the brownish polymer conjugate precipitatesout of solution. The polymer is then washed with IPA (2×20 mL) to removeresidual reagents and dried under vacuum at 35-40° C. for 2 days as abrownish powder (1.0 g).

Example 28: Preparation of PLGA-2,9-Dibenzyl-8-Hydroxyadenine Conjugate

A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrousEtOAc (30 mL) is stirred at RT under argon for 30 minutes. Compound (II)(0.24 g, 0.7 mmol) in 2 mL of dry DMSO is added, followed bydiisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). The mixture isstirred at RT for 20 h. Additional amounts of HBTU (0.53 g, 1.4 mmol)and DIPEA (0.5 mL, 2.8 mmol) are added and the mixture is heated at50-55° C. for 4 h. After cooling, the mixture is diluted with EtOAc (100mL) and washed with saturated NH₄Cl solution 20 mL), water (2×20 mL) andbrine solution (20 mL). The solution is dried over Na₂SO₄ (10 g) andconcentrated to a gel-like residue. Isopropyl alcohol (IPA) (35 mL) isthen added and the brownish polymer conjugate precipitated out ofsolution. The polymer is then washed with IPA (2×20 mL) to removeresidual reagents and dried under vacuum at 35-40° C. for 2 days as abrownish powder (1.2 g).

Example 29: Imide Ring Opening Used to Attach2-Pentyl-8-Hydroxy-9-Benzyladenine to the Terminal Alcohol Groups ofPoly-Hexamethylene Carbonate) Diol of Molecular Weight 2000

The poly(hexamethylene carbonate) diol is purchased from AldrichChemical Company, Cat #461164.

Poly(hexamethylene carbonate) diol:HO—[CH₂(CH₂)₄CH₂OCO₂ ]nCH₂(CH₂)₄CH₂—OH

Poly(hexamethylene carbonate) diol-8-oxoadenine conjugate:

The polymer (5 g, 2.5×10⁻³ moles) is dissolved in methylene chloride 25mL and the lactam of 2-pentyl-8-hydroxy-9-benzyladenine (2.05 g,5.0×10⁻³ moles) is added. This slurry is stirred as1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4×10⁻³ moles) isadded in a single portion. After stirring at room temperature overnighta clear pale yellow solution forms. The solution is diluted withmethylene chloride (100 mL), and the solution is washed with 5% citricacid. This solution is dried over sodium sulfate after which it isfiltered and evaporated under vacuum. After drying under high vacuumthere is obtained 5.5 grams (78%) of polymer. NMR is used to determinethe benzyladenine content which is 18%.

Example 30: Nicotine-Peg-Pla Conjugates

A 3-nicotine-PEG-PLA polymer was synthesized as follows:

First, monoamino poly(ethylene glycol) from JenKem® with a molecularweight of 3.5 KD (0.20 g, 5.7×10⁻⁵ moles) and an excess of4-carboxycotinine (0.126 g, 5.7×10⁻⁴ moles) were dissolved indimethylformamide (5.0 mL). The solution was stirred anddicyclohexylcarbodiimide (0.124 g, 6.0×10⁻⁴ moles) was added. Thissolution was stirred overnight at room temperature. Water (0.10 mL) wasadded and stirring was continued for an additional 15 minutes. Theprecipitate of dicyclohexyl urea was removed by filtration and thefiltrates were evaporated under vacuum. The residue was dissolved inmethylene chloride (4.0 mL) and this solution was added to diethyl ether(100 mL). The solution was cooled in the refrigerator for 2 hours andthe precipitated polymer was isolated by filtration. After washing withdiethyl ether, the solid white polymer was dried under high vacuum. Theyield was 0.188 g. This polymer was used without further purificationfor the next step.

The nicotine/PEG polymer (0.20 g, 5.7×10⁻⁵ moles) was dissolved in drytetrahydrofuran (10 mL) under nitrogen and the solution was stirred as asolution of lithium aluminum hydride in tetrahydrofuran (1.43 mL of 2.0M, 2.85×10⁻³ moles) was added. The addition of the lithium aluminumhydride caused the polymer to precipitate as a gelatinous mass. Thereaction was heated to 80° C. under a slow stream of nitrogen and thetetrahydrofuran was allowed to evaporate. The residue was then heated at80° C. for 2 hours. After cooling, water (0.5 mL) was cautiously added.Once the hydrogen evolution had stopped, 10% methanol in methylenechloride (50 mL) was added and the reaction mixture was stirred untilthe polymer had dissolved. This mixture was filtered through Celite®brand diatomaceous earth (available from EMD Inc. as Celite® 545, part#CX0574-3) and the filtrates were evaporated to dryness under vacuum.The residue was dissolved in methylene chloride (4.0 mL) and thissolution was slowly added to diethyl ether (100 mL). The polymerseparated as a white flocculent solid and was isolated bycentrifugation. After washing with diethyl ether, the solid was driedunder vacuum. The yield was 0.129 g.

Next, a 100 mL round bottom flask, equipped with a stir bar and refluxcondenser was charged with the PEG/nicotine polymer (0.081 g, 2.2×10⁻⁵moles), D/L lactide (0.410 g, 2.85×10⁻³ moles) and anhydrous sodiumsulfate (0.380 g). This was dried under vacuum at 55° C. for 8 hours.The flask was cooled and flushed with argon and then dry toluene (10 mL)was added. The flask was placed in an oil bath set at 120° C., and oncethe lactide had dissolved, tin ethylhexanoate (5.5 mg, 1.36×10⁻⁵ moles)was added. The reaction was allowed to proceed at 120° C. for 16 hours.After cooling to room temperature, water (15 mL) was added and stirringwas continued for 30 minutes. Methylene chloride (200 mL) was added, andafter agitation in a separatory funnel, the phases were allowed tosettle. The methylene chloride layer was isolated and dried overanhydrous magnesium sulfate. After filtration to remove the dryingagent, the filtrates were evaporated under vacuum to give the polymer asa colorless foam. The polymer was dissolved in tetrahydrofuran (10 mL)and this solution was slowly added to water (150 mL) with stirring. Theprecipitated polymer was isolated by centrifugation and the solid wasdissolved in methylene chloride (10 mL). The methylene chloride wasremoved under vacuum and the residue was dried under vacuum.3-nicotine-PEG-PLA polymer yield was 0.38 g

Example 31: Synthetic Nanocarrier Formulation

For encapsulated adjuvant formulations, Resiquimod (aka R848) wassynthesized according to the synthesis provided in Example 99 of U.S.Pat. No. 5,389,640 to Gerster et al.

R848 was conjugated to PLA by a method provided above, and the PLAstructure was confirmed by NMR.

PLA-PEG-nicotine conjugate was prepared according to Example 30.

PLA was purchased (Boehringer Ingelheim Chemicals, Inc., 2820 NorthNormandy Drive, Petersburg, Va. 23805). The polyvinyl alcohol (Mw=11KD-31 KD, 85-89% hydrolyzed) was purchased from VWR scientific.Ovalbumin peptide 323-339 was obtained from Bachem Americas Inc. (3132Kashiwa Street, Torrance Calif. 90505. Part #4064565).

The above materials were used to prepare the following solutions:

1. Resiquimod (R848) @ 10 mg/mL and PLA @ 100 mg/mL in methylenechloride or PLA-R848 conjugate @ 100 mg/mL in methylene chloride

2. PLA-PEG-nicotine in methylene chloride @ 100 mg/mL

3. PLA in methylene chloride @ 100 mg/mL

4. Ovalbumin peptide 323-339 in water @ 10 or 69 mg/mL

5. Polyvinyl alcohol in water @ 50 mg/mL.

Solution #1 (0.25 to 0.75 mL), solution #2 (0.25 mL), solution #3 (0.25to 0.5 mL) and solution #4 (0.1 mL) were combined in a small vial andthe mixture was sonicated at 50% amplitude for 40 seconds using aBranson Digital Sonifier 250. To this emulsion was added solution #5(2.0 mL) and sonication at 35% amplitude for 40 seconds using theBranson Digital Sonifier 250 forms the second emulsion. This was addedto a beaker containing phosphate buffer solution (30 mL) and thismixture was stirred at room temperature for 2 hours to form thenanoparticles.

To wash the particles a portion of the nanoparticle dispersion (7.4 mL)was transferred to a centrifuge tube and spun at 5,300 g for one hour,supernatant was removed, and the pellet was re-suspended in 7.4 mL ofphosphate buffered saline. The centrifuge procedure was repeated and thepellet was re-suspended in 2.2 mL of phosphate buffered saline for afinal nanoparticle dispersion of about 10 mg/mL.

Example 32: Double Emulsion with Multiple Primary Emulsions

Materials

Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T cellepitope of Ovalbumin protein, was purchased from Bachem Americas Inc.(3132 Kashiwa Street, Torrance Calif. 90505.)

Resiquimod (aka R848) was synthesized according to a method provided inU.S. Pat. No. 6,608,201.

PLA-R848, resiquimod, was conjugated to PLA with a molecular weight ofapproximately 2,500 Da according to a method provided above.

PLGA-R848, resiquimod, was conjugated to PLGA with a molecular weight ofapproximately 4,100 Da according to a method provided above.

PS-1826 DNA oligonucleotide with fully phosphorothioated backbone havingnucleotide sequence 5′-TCC ATG ACG TTC CTG ACG TT-3′ (SEQ ID NO: 1) witha sodium counter-ion was purchased from Oligos Etc (9775 SW CommerceCircle C-6, Wilsonville, Oreg. 97070.)

PO-1826 DNA oligonucleotide with phosphodiester backbone havingnucleotide sequence 5′-TCC ATG ACG TTC CTG ACG TT-3′ (SEQ ID NO: 2) witha sodium counter-ion was purchased from Oligos Etc. (9775 SW CommerceCircle C-6, Wilsonville, Oreg. 97070.)

PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModicsPharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211. ProductCode 100 DL 2A.)

PLA with an inherent viscosity of 0.71 dL/g was purchased from SurModicsPharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211. ProductCode 100 DL 7A.)

PLA with an inherent viscosity of 0.19 dL/g was purchased fromBoehringer Ingelheim Chemicals, Inc. (Petersburg, Va. Product CodeR202H.)

PLA-PEG-nicotine with a molecular weight of approximately 18,500 to22,000 Da was prepared according to a method provided above.

PLA-PEG-R848 with a molecular weight of approximately 15,000 Da wassynthesized was prepared according to a method provided above.

Polyvinyl alcohol (Mw=11,000-31,000, 87-89% hydrolyzed) was purchasedfrom J. T. Baker (Part Number U232-08).

Batches were produced using a double emulsion process with multipleprimary emulsions. The table below references the solution suffix (e.g.,B in Solution #1 column indicates Solution #1B was used) and volume ofsolution used.

Solution Solution Solution Sample #1 #2 #3 Solution #4 Solution #5Number (Volume) (Volume) (Volume) (Volume) (Volume) 1 B (0.1 ml) C (1.0ml) A (0.1 ml) C (1.0 ml) A (2.0 ml) 2 A (0.2 ml) A (1.0 ml) A (0.1 ml)A (1.0 ml) A (3.0 ml) 3 A (0.2 ml) B (1.0 ml) A (0.1 ml) B (1.0 ml) A(3.0 ml) 4 A (0.2 ml) B (1.0 ml) A (0.1 ml) B (1.0 ml) A (3.0 ml)

Solution 1A: Ovalbumin peptide 323-339 @ 35 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13N hydrochloric acid solution at room temperature.

Solution 1B: Ovalbumin peptide 323-339 @ 70 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13N hydrochloric acid solution at room temperature.

Solution 2A: 0.21-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml inmethylene chloride. The solution was prepared by first preparing twoseparate solutions at room temperature: 0.21-IV PLA @ 100 mg/mL in puremethylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure methylenechloride. The final solution was prepared by adding 3 parts PLA solutionfor each part of PLA-PEG-nicotine solution.

Solution 2B: 0.71-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml inmethylene chloride. The solution was prepared by first preparing twoseparate solutions at room temperature: 0.71-IV PLA @ 100 mg/mL in puremethylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure methylenechloride. The final solution was prepared by adding 3 parts PLA solutionfor each part of PLA-PEG-nicotine solution.

Solution 2C: 0.19-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml inmethylene chloride. The solution was prepared by first preparing twoseparate solutions at room temperature: 0.19-IV PLA @ 100 mg/mL in puremethylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure methylenechloride. The final solution was prepared by adding 3 parts PLA solutionfor each part of PLA-PEG-nicotine solution.

Solution 3A: Oligonucleotide (either PS-1826 or P0-1826) @ 200 mg/ml inpurified water. The solution was prepared by dissolving oligonucleotidein purified water at room temperature.

Solution 4A: Same as Solution #2A.

Solution 4B: Same as Solution #2B.

Solution 4C: Same as Solution #2C.

Solution 5A: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

Two separate primary water in oil emulsions were prepared. W1/O2 wasprepared by combining solution 1 and solution 2 in a small pressure tubeand sonicating at 50% amplitude for 40 seconds using a Branson DigitalSonifier 250. W3/O4 was prepared by combining solution 3 and solution 4in a small pressure tube and sonicating at 50% amplitude for 40 secondsusing a Branson Digital Sonifier 250. A third emulsion with two inneremulsion ([W1/O2,W3/O4]/W5) emulsion was prepared by combining 0.5 ml ofeach primary emulsion (W1/O2 and W3/O4) and solution 5 and sonicating at30% amplitude for 40 to 60 seconds using the Branson Digital Sonifier250.

The third emulsion was added to a beaker containing 70 mM phosphatebuffer solution (30 mL) and stirred at room temperature for 2 hours toallow for the methylene chloride to evaporate and for the nanocarriersto form. A portion of the nanocarriers were washed by transferring thenanocarrier suspension to a centrifuge tube and spinning at 13,823 g forone hour, removing the supernatant, and re-suspending the pellet inphosphate buffered saline. The washing procedure was repeated and thepellet was re-suspended in phosphate buffered saline for a finalnanocarrier dispersion of about 10 mg/mL.

The amounts of oligonucleotide and peptide in the nanocarrier weredetermined by HPLC analysis.

Example 33: Standard Double Emulsion

Materials

As provided in Example 32 above.

Batches were produced using a standard double emulsion process. Thetable below references the solution suffix (e.g., B in Solution #1column indicates Solution #1B was used) and volume of solution used.

Sam- ple Solution Solution Num- #1 Solution #2 Solution #3 Solution #4#5 ber (Volume) (Volume) (Volume) (Volume) (Volume) 1 A (0.1 ml) A (0.75ml) A (0.25 ml) None A (2.0 ml) 2 A (0.1 ml) None A (0.25 ml) A (0.75ml) A (2.0 ml) 3 A (0.1 ml) B (0.75 ml) A (0.25 ml) None A (2.0 ml) 4 B(0.1 ml) C (0.75 ml) A (0.25 ml) None B (2.0 ml) 5 B (0.1 ml) D (0.25ml) A (0.25 ml) A (0.50 ml) B (2.0 ml) 6 C (0.2 ml) None A (0.25 ml) A(0.75 ml) B (2.0 ml) 7 D (0.1 ml) None A (0.25 ml) A (0.75 ml) B (2.0ml)

Solution 1A: Ovalbumin peptide 323-339 @ 69 mg/mL in de-ionized water.The solution was prepared by slowly adding ovalbumin peptide to thewater while mixing at room temperature.

Solution 1B: Ovalbumin peptide 323-339 @ 70 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13N hydrochloric acid solution at room temperature.

Solution 1C: Oligonucleotide (PS-1826) @ 50 mg/ml in purified water. Thesolution was prepared by dissolving oligonucleotide in purified water atroom temperature.

Solution 1D: Ovalbumin peptide 323-339 @ 17.5 mg/mL in dilutehydrochloric acid aqueous solution. The solution was prepared bydissolving ovalbumin peptide @ 70 mg/ml in 0.13N hydrochloric acidsolution at room temperature and then diluting the solution with 3 partspurified water per one part of starting solution.

Solution 2A: R848 @ 10 mg/ml and 0.19-IV PLA @ 100 mg/mL in puremethylene chloride prepared at room temperature.

Solution 2B: PLA-R848 @ 100 mg/ml in pure methylene chloride prepared atroom temperature.

Solution 2C: PLGA-R848 @ 100 mg/ml in pure methylene chloride preparedat room temperature.

Solution 2D: PLA-PEG-R848 @ 100 mg/ml in pure methylene chlorideprepared at room temperature.

Solution 3A: PLA-PEG-nicotine @ 100 mg/ml in pure methylene chlorideprepared at room temperature.

Solution 4A: 0.19-IV PLA @ 100 mg/mL in pure methylene chloride preparedat room temperature.

Solution 5A: Polyvinyl alcohol @ 50 mg/mL in de-ionized water.

Solution 5B: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

The water in oil (W/O) primary emulsion was prepared by combiningsolution 1 and solution 2, solution 3, and solution 4 in a smallpressure tube and sonicating at 50% amplitude for 40 seconds using aBranson Digital Sonifier 250. The water/oil/water (W/O/W) doubleemulsion was prepared by adding solution 5 to the primary emulsion andsonicating at 30% to 35% amplitude for 40 seconds using the BransonDigital Sonifier 250.

The double emulsion was added to a beaker containing phosphate buffersolution (30 mL) and stirred at room temperature for 2 hours to allowfor the methylene chloride to evaporate and for the nanocarriers toform. A portion of the nanocarriers were washed by transferring thenanocarrier suspension to a centrifuge tube and spinning at 5,000 to9,500 RPM for one hour, removing the supernatant, and re-suspending thepellet in phosphate buffered saline. The washing procedure was repeatedand the pellet was re-suspended in phosphate buffered saline for a finalnanocarrier dispersion of about 10 mg/mL.

Example 34: Determination of Amount of Agents

Method for R848 and Peptides (e.g., Ova Peptide, Human Peptide,TT2pDT5t)

The amount of R848 (immunostimulatory agent) and ova peptide (T cellantigen) was measured using reverse phase HPLC on an Agilent 1100 systemat appropriate wavelengths (λ=254 nm for R848 and 215 nm for ovapeptide) equipped with an Agilent Zorbax SB-C18 column (3.5 μm. 75×4.6mm. Column Temp=40° C. (part no. 866953-902)) using Mobile Phase A (MPA)of 95% water/5% acetonitrile/0.1% TFA and Mobile Phase B (MPB) of 90%acetonitrile/10% water/0.09% TFA (Gradient: B=5 to 45% in 7 minutes;ramp to 95% B to 9 min; decrease back to 5% B to 9.5 min and keptequilibrating to end. Total run time was 13 minute with flow rate of 1mL/min).

Method for CpG

The amount of CpG (immunostimulatory agent) was measured using reversephase HPLC on Agilent 1100 system at 260 nm equipped with Waters XBridgeC-18 (2.5 micron particle, 50×4.6 mm ID (part No. 186003090), columntemp. 600C) using mobile phase A of 2% acetonitrile in 100 mM TEA-aceticacid buffer, pH about 8.0 and mobile B as 90% acetonitrile, 10% water(column equilibrated at 5% B, increased to 55% B in 8.5 min, then rampedto 90% B to 12 minutes. Strength of B was rapidly decreased to 5% in oneminute and equilibrated until stop time, 16 minutes. The flow rate was 1mL/min until end of the method, 16 minutes).

Method for Nicotine Analog

Nicotine analog was measured using reverse phase HPLC on Agilent 1100system at 254 nm equipped with Waters X-Bridge C-18 (5 micron particle,100×4.6 mm ID, column temp at 400 C) using Mobile Phase A (MPA) of 95%water/5% acetonitrile/0.1% TFA and Mobile Phase B (MPB) of 90%acetonitrile/10% water/0.09% TFA (gradient: column was equilibrated at5% B increased to 45% B in 14 minutes. Then ramped up to 95% B from 14to 20 minutes. Mobile B strength was quickly decreased back to 5% andrequilibrated until the end of the method. The flow rate of the methodwas maintained at 0.5 ml/min with total run time of 25 minutes. The NCsuspension was centrifuged @ 14000 rpm for about 15-30 minutes dependingon particle size. The collected pellets were treated with 200 uL ofconc. NH₄OH (8 M) for 2 h with agitation until the solution turns clear.A 200 uL of 1% TFA was added to neutralize the mixture solution, whichbrought the total volume of the pellet solution to 200 uL. An aliquot of50 uL of the solution was diluted with MPA (or water) to 200 uL andanalyzed on HPLC as above to determine the amount present in thepellets.

Encapsulated Free R848 in Nanocarrier

0.5 mL of the NC suspension was centrifuged @ 14000 rpm for about 15minutes. The collected pellet was dissolved with 0.3 mL of acetonitrileand centrifuged briefly @ 14000 rpm to remove any residual insolubles.The clear solution was further diluted with 4 times equivalent volume ofMPA and assayed on reverse phase HPLC described above.

Encapsulated CpG in Nanocarrier

330 uL of NC suspension from the manufacture (about 10 mg/mL suspensionin PBS) was spun down at 14000 rpm for 15 to 30 minutes depending onparticle size. The collected pellets were re-suspended with 500 uL ofwater and sonicated for 30 minutes to fully disperse the particles. TheNC was then heated at 600° C. for 10 minutes. Additional 200 uL of 1 NNaOH was added to the mixture, heated for another 5 minutes where themixture becomes clear. The hydrolyzed NC solution was centrifugedbriefly at 14000 rpm. A final 2× dilution of the clear solution usingwater was then made and assayed on the reverse HPLC described above.

Encapsulated T Cell Antigens (e.g., Ova Peptide, or Human Peptide,TT2pDT5t)

330 uL of NC suspension from the manufacture (about 10 mg/mL suspensionin PBS) was spun down at 14000 rpm for 15 to 30 minutes. 100 uL ofacetonitrile was added to the pellets to dissolve the polymer componentsof the NC. The mixture was vortexed and sonicated for 1 to 5 minutes.100 uL 0.2% TFA was added to the mixture to extract the peptides andsonicated for another 5 minutes to ensure the break down of theaggregates. The mixture was centrifuged at 14000 rpm for 15 minutes toseparate any insoluble materials (e.g., polymers). A 50 uL aliquot ofthe supernatant diluted with 150 uL of MPA (or water) was taken andassayed on the reverse phase HPLC as described above.

Amount of Conjugated Nicotine Analog (B Cell Antigen) in Nanocarriers

1.5 mL of NC suspension was spun down @ 14000 rpm for about 15 minutes,the pellets were hydrolyzed using 150 uL of concentrated NH₄OH (8M) forabout 2-3 h until the solution turns clear. A 150 uL of 2% TFA(aq)solution was added to the pellet mixture to neutralize the solution. A100 uL aliquot of the mixture was diluted with 200 uL of water andassayed on reverse phase HPLC described above and quantified based onthe standard curve established using the precursor (PEG-nicotine) of thePLA-PEG-nicotine used in the manufacture.

Example 35: Release Rate of Immunomodulatory Agent from SyntheticNanocarriers

The following data show the rates of release of R-848 from nanoparticlesmade from the low molecular weight polylactic acid-R-848 conjugate shownabove. Table 1 provides relevant formulation information for theexperiments.

The release of T-cell antigen, ova peptide and adjuvant, R848 from thesynthetic nanocarrier (nanoparticles) in PBS (100 mM, pH=7.4) andCitrate buffer (100 mM, pH=4.5) at 37° C. were performed as follows:

Analytical Method: The amount of R848 and ova peptide released ismeasured using reverse phase HPLC on a Agilent 1100 system at λ=215 nmequipped with an Agilent Zorbax SB-C18 column (3.5 μm. 75×4.6 mm. ColumnTemp=40° C. (part no. 866953-902)) using Mobile Phase A (MPA) of 98%water/2% acetonitrile/0.1% TFA and Mobile Phase B (MPB) of 90%acetonitrile/10% water/0.09% TFA with Gradient: B=5 to 45% in 7 minutes;ramp to 95% B to 9 min; re-EQ to end. 13 minute run time. Flow=1 mL/min.

The total amount of R848 and ova peptide present in the nanoparticleswas as shown in Table 1. An aqueous suspension of the tested syntheticnanocarriers was then diluted to a final stock volume of 4.4 mL withPBS.

(A) In Vitro Release Rate Measurement in PBS (pH=7.4):

For T0 sample, a 200 μL aliquot was immediately removed from each of theNP sample and centrifuged @ 14000 rpm in a microcentrifuge tubes using aMicrocentrifuge (Model: Galaxy 16). 100 μL of supernatant was removedand diluted to 200 μL in HPLC Mobile Phase A (MPA) and assayed for theamount of R848 and ova peptide released on the reverse phase HPLC.

For time point measurements: 9×200 μL of each of the samples were addedto microcentrifuge tubes (3×200 for unconjugated) and 300 μL of 37 C PBSwas added to each above aliquot and the samples were placed immediatelyin 37° C. oven. At the following time points: 24 hr, 48 hr, 96 hr and144 hr (for conjugated R848) or 2 h, 16 h and 24 h (for unconjugated(encapsulated) R848), the samples were centrifuged and assayed for theamount of R848 and ova peptide released as above for T0 sample.

(B) In Vitro Release Rate Measurement in Citrate Buffer (pH=4.5):

For T0 sample, a 200 μL aliquot was removed from each of the samples andcentrifuged @ 6000 rpm for 20 minutes and the supernatant was removed.The residue nanoparticles was resuspended in 200 uL of citrate bufferand centrifuged @ 14000 rpm for 15 minutes. 100 uL of the supernatantwas removed and diluted to 200 uL with MPA and assayed for R848 andpeptide as above.

For time point measurements: 9×200 uL of each of the samples were addedto microcentrifuge tubes (3×200 for unconjugated) and centrifuged for 20minutes @ 6000 rpm and the supernatants were removed. The residue NPswere then resuspended in 500 uL of citrate buffer and placed in 37° C.oven. At the following time points: 24 hr, 48 hr, 96 hr and 144 hr (forconjugated R848) or 2 h, 16 h and 24 h (for unconjugated (encapsulated)R848), the samples were centrifuged and assayed for the amount of R848and ova peptide released as above for T0 sample.

In order to complete the mass balance from above measurements in PBS andCitrate buffer, the remaining pellets (conjugated R848 samples only)from each sample was treated with 200 uL of conc. NH4OH (8 M) for 3 hwith mixing. After the mixture was settled, 200 uL of 1% TFA was addedto bring total volume of the pellet to 400 uL. An aliquot of 50 uL ofthe solution was diluted with MPA to 200 uL and analyzed on HPLC asabove to determine the amount of R848 and ova peptide that remained inthe pellet after in vitro release to close the mass balance. Forunconjugated samples, the sample was diluted with TFA in acetonitrileand assayed as above for R848 and peptide.

The results are summarized in FIGS. 1-3.

TABLE 1 Formulation Targets With A Covalent R848 PLA- R848 PLA For- OvaPLA- conju- (15- mula- R848 peptide PEG- gate 20K, BI Chem- tion load*load NIC type** R202H) istry 1 E1.5%   1.1-2.2% 25% 75% 2   E1.5%++ 1.1-2.2% 25% 75% 3 C75% 0.15-0.31% 25% Method 1 Amine 4 C75% 0.15-0.31%25% Method 1 Amine 5 C75% 0.15-0.31% 25% Method 5 ROP-hi MW 6 C75%0.15-0.31% 25% Method 5 ROP-lo MW 7 C50% 0.15-0.31% 25% Method 5 25%ROP-lo MW 8 C25% 0.15-0.31% 25% Method 5 50% ROP-lo MW *C = covalentR848; E = encapsulation of R848Materials and Method—

-   -   HPLC—Agilent 1100. λ=215 nm. Column Temp=40° C.    -   Column—Agilent Zorbax SB-C18, 3.5 μm. 75×4.6 mm. (part no.        866953-902) C18 guard column    -   Mobile Phase A (MPA)—98% water/2% acetonitrile/0.1% TFA    -   Mobile Phase B (MPB)—90% acetonitrile/10% water/0.09% TFA        -   Gradient: B=5 to 45% in 7 minutes; ramp to 95% B to 9 min;            re-EQ to end. 13 minute run time. Flow=1 mL/min.    -   PBS—100 mM, pH=7.4.    -   Citrate Buffer—100 mM, pH=4.5.    -   Oven—    -   Microcentrifuge—Galaxy 16    -   Microcentrifuge tubes    -   Sonicator    -   Pipets—20, 200, 1000 μL adjustable    -   HPLC grade water—EMD-#WX0008-1.    -   NH₄OH—˜8M. Mallinkcrodt.    -   TFA, 0.2%. Prep 4/27/09.    -   TFA, 1%. Prep 5/13/09.    -   Thermometer        Samples—

“6-1” and “6-2” have entrapped R848. All of the rest have conjugatedR848.

The estimated values are based on the loading results from the “62”series.

TABLE 2 Estimated R848 and Ova peptide in synthetic nanocarriers:Estimated R848 in Estimated Ova in Sample ID NPs (μg/mL) NPs (μg/mL) 154 146 2 166 184 3 119 32 4 114 34 5 465 37 6 315 34 7 116 40

Sample volumes were slightly below what was planned. To ensure enoughmaterial is available for all time points, the following volumes of PBSwere added to the samples to bring them all to 4.4 mL.

TABLE 3 Sample Volume PBS added Sample ID Volume (mL) (mL) 1 4.35 0.05 24.23 0.17 3 4.21 0.19 4 4.20 0.20 5 4.21 0.19 6 4.19 0.21 7 4.20 0.20Procedure—

-   -   1) T=0 Sample Prep        -   a. PBS            -   i. Remove a 200 μL aliquot from each of the samples.                Microcentrifuge @ 14000 rpm. Remove supernatant.            -   ii. Dilute supernatant 100 μL>200 μL in MPA. (DF=2).            -   iii. Assay for peptide and R848.        -   b. Citrate            -   i. Remove a 200 μL aliquot from each of the samples.                Microcentrifuge @ 6000 rpm for 20 minutes. Remove                supernatant.            -   ii. Add 200 uL of citrate buffer and thoroughly                resuspend.            -   iii. Microcentrifuge @ 14000 rpm for 15 minutes. Remove                supernatant.            -   iv. Dilute supernatant 100 μL >200 μL in MPA. (DF=2)            -   v. Assay for peptide and R848.    -   2) PBS IVR        -   a. Add 9×200 μL of each of the samples to microcentrifuge            tubes. (3×200 for unconjugated)        -   b. To each aliquot add 300 μL of 37 C PBS.        -   c. Immediately place samples in 37 C oven.    -   3) Citrate IVR        -   a. Add 9×200 uL of each of the samples to microcentrifuge            tubes. (3×200 for unconjugated)        -   b. Centrifuge for 20 minutes @ 6000 rpm.        -   c. Remove the supernatants.        -   d. To each tube, add 500 μL of citrate buffer and resuspend            thoroughly.        -   e. Place samples in 37 C oven    -   4) For lots 1-4 and 8, remove the samples (see step 6) at the        following time points:        -   a. Conjugated            -   i. 24 hr            -   ii. 48 hr (2 days)            -   iii. 96 hr (4 days)            -   iv. 144 hr (6 days)            -   v. Further time points TBD based on the above data.        -   b. Non conjugated            -   i. 2 hr            -   ii. 16 hr            -   iii. 24 hr    -   5) For lots 6 and 7, remove samples at the following time        points:        -   a. PBS            -   i. 24 hr            -   ii. 48 hr (2 days)            -   iii. 96 hr (4 days)            -   iv. 144 hr (6 days)            -   v. Further time points TBD based on the above data.        -   b. Citrate            -   i. 2 hr            -   ii. 16 hr            -   iii. 24 hr            -   iv. 48 hr (2 days)            -   v. 72 hr (3 days)            -   vi. 96 hr (4 days)            -   vii. 120 hr (5 days)            -   viii. Further time points TBD based on the above data.    -   6) Sample as follows:        -   a. Microcentrifuge @ 14000 rpm for 15 minutes.        -   b. Remove supernatant.        -   c. Dilute 100 μL to 200 μL in MPA. (DF=2)    -   7) Assay for peptide and R848. This will provide the amount        released at each time point.

To Complete Mass Balance, Perform the Following:

-   -   8) To the remaining pellets (conjugated only) add 200 uL NH₄OH.    -   9) Vortex briefly and sonicate to disperse.    -   10) Add stir bar. Allow to sit until clear (at least 3 hours).    -   11) Add 200 uL of 1% TFA (total pellet volume=400 μL).    -   12) Dilute 50 μL to 200 μL in MPA. Analyze by HPLC to determine        peptide and R848 remaining in the pellet. (DF=4).    -   13) For unconjugated lots, assay for peptide and R848 with        typical AcN/TFA method.

Example 36: Release Rate Testing

The release of antigen (e.g., ova peptide, T cell antigen) andimmunostimulatory agents (e.g., R848, CpG) from synthetic nanocarriersin phosphate buffered saline solution (PBS) (100 mM, pH=7.4) and citratebuffer (100 mM, pH=4.5) at 37° C. was determined as follows:

The release of R848 from the nanocarrier composed of conjugated R848 andthe ova peptide was achieved by exchanging desired amount of the aqueoussuspension of the tested synthetic nanocarriers obtained from themanufacture (e.g., about 10 mg/mL in PBS) into the same volume of theappropriate release media (Citrate buffer 100 mM) via centrifugation andre-suspension.

In Vitro Release Rate Measurement in PBS (pH=7.4)

1 mL of the PBS suspension NC was centrifuged @ 14000 rpm inmicrocentrifuge tubes generally from 15-30 minutes depending on particlesize. The collected supernatant was then diluted with equal volume ofthe mobile phase A (MPA) or water and assayed on reverse phase HPLC forthe amount of the R848 release during the storage. The remaining pelletwas re-suspended to homogeneous suspension in 1 mL of PBS and placed to37° C. thermal chamber with constant gentle agitation

For T0 sample, a 150 μL aliquot was immediately removed from NCsuspension prior placing the NC suspension to 37° C. thermal chamber andcentrifuged @ 14000 rpm in microcentrifuge tubes using a microcentrifuge(Model: Galaxy 16). 100 μL of the supernatant was removed and diluted to200 μL with HPLC Mobile Phase A (MPA) or water and assayed for theamount of R848 and ova peptide released on the reverse phase HPLC.

For time point measurements, 150 μL aliquot was removed from the 37° C.NC sample suspension, and the samples were centrifuged and assayed forthe amount of R848 and ova peptide released in the same manner as for T0sample. The R848 and ova peptide released was tested at 6 h, 24 h forroutine monitoring with additional 2 h, 48 h, 96 h and 144 h forcomplete release profile establishment.

In Vitro Release Rate Measurement in Citrate Buffer (pH=4.5)

A 100 mM sodium citrate buffer (pH=4.5) was applied to exchange theoriginal NC storage solution (e.g., PBS) instead of the PBS buffer,pH=7.4. In order to complete the mass balance from above measurements inPBS and Citrate buffer, the remaining pellets from each time point weretreated with 100 uL of NH₄OH (8 M) for 2 h (or more) with agitationuntil solution turn clear. A 100 uL of 1% TFA was added to neutralizethe mixture, which brought the total volume of the pellet solution to200 uL. An aliquot of 50 uL of the mixture was diluted with MPA (orwater) to 200 uL and analyzed on HPLC as above to determine the amountof unreleased R848 remaining in the pellets after in vitro release toclose the mass balance. For unconjugated samples, the sample was dilutedwith TFA in acetonitrile and assayed as above for R848.

The release of CpG was determined similar to the measurement of R848 andova peptide in terms of sample preparation and monitored time points.However, the amount of the CpG in the release media was assayed by thereverse phase HPLC method described above.

Example 37: Immunization with NC-Nic Carrying CpG Adjuvant

Groups of five mice were immunized three times (subcutaneously, hindlimbs) at 2-week intervals (days 0, 14 and 28) with 100 μg of NC-Nic.NC-Nic was a composition of nanocarriers exhibiting nicotine on theouter surface and, for all groups of mice except for Group 1, carryingCpG-1826 (thioated) adjuvant, which was released from the nanocarriersat different rates. The nanocarriers were prepared according to a methodprovided above. Serum anti-nicotine antibodies were then measured ondays 26 and 40. EC₅₀ for anti-nicotine antibodies as measured instandard ELISA against polylysine-nicotine are shown in FIG. 4.

The Group 1 mice were administered NC-Nic w/o CpG-1826 containing Ovapeptide and polymers, 75% of which were PLA and 25% were PLA-PEG-Nic.The Group 2 mice were administered NC-Nic containing ova peptide,polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 3.2%CpG-1826; release rate at 24 hours: 4.2 μg CpG per mg of NC. The Group 3mice were administered NC-Nic containing polymers, 75% of which were PLAand 25% were PLA-PEG-Nic, and 3.1% CpG-1826; release rate at 24 hours:15 μg CpG per mg of NC. Release was determined at a pH of 4.5.

The results shown in FIG. 4 demonstrate that entrapment of adjuvant intonanocarriers is beneficial for the immune response against NC-associatedantigen, and, furthermore, that the higher release rate of entrapped CpGadjuvant from within the nanocarriers (NC) at 24 hours produced animmune response, which was elevated compared to one induced by NC with aslower release rate of CpG adjuvant (a TLR9 agonist).

Example 38: Immunization with NC-Nic Carrying Two Forms of CpG Adjuvant

Groups of five mice were immunized two times (subcutaneously, hindlimbs) at 4-week intervals (days 0, and 28) with 100 μg of NC-Nic andserum anti-nicotine antibodies were then measured on days 12, 24 and 40.NC-Nic was a composition of nanocarriers exhibiting nicotine on theouter surface and carrying one of two forms of CpG-1826 adjuvant. Thenanocarriers were prepared according to a method provided above. EC₅₀for anti-nicotine antibodies as measured in standard ELISA againstpolylysine-nicotine are shown in FIG. 5.

The Group 1 mice were administered NC-Nic containing ova peptide,polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 6.2%CpG-1826 (thioated); release rate at 24 hours: 16.6 μg CpG per mg of NC.The Group 2 mice were administered NC-Nic containing ova peptide,polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 7.2%CpG-1826 (thioated); release rate at 24 hours: 13.2 μg CpG per mg of NC.The Group 3 mice were administered NC-Nic containing ova peptide,polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 7.9%CpG-1826 (phosphodiester or PO, non-thioated); release rate at 24 hours:19.6 μg CpG per mg of NC. The Group 4 mice were administered NC-Niccontaining ova peptide, polymers, 75% of which were PLA and 25% werePLA-PEG-Nic, and 8.5% CpG-1826 (PO, non-thioated); release rate at 24hours: 9.3 μg CpG per mg of NC. Release was determined at a pH of 4.5.

The results shown in FIG. 5 demonstrate that the rate of release ofentrapped adjuvant (CpG, TLR9 agonist) from nanocarriers influencedproduction of an antibody to NC-bound antigen (nicotine) with thenanocarrier exhibiting higher release rate at 24 hours induced strongerhumoral immune response (group 1>group 2 and group 3>group 4). This wastrue irrespective of CpG form used (more stable, thioated or less stablenon-thioated).

Example 39: Immunization with NC-Nic Carrying R848

Groups of five mice were immunized three times (subcutaneously, hindlimbs) at 2-week intervals (days 0, 14 and 28) with 100 μg of NC-Nic andserum anti-nicotine antibodies were then measured on days 26, 40 and 54.The nanocarriers were prepared according to a method provided above.EC₅₀ for anti-nicotine antibodies as measured in standard ELISA againstpolylysine-nicotine are shown in FIG. 6.

The Group 1 mice were administered NC-Nic containing ova peptide andpolymers, 75% of which were PLA and 25% were PLA-PEG-Nic, but withoutadjuvant. The Group 2 mice were administered NC-Nic containing ovapeptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and1.0% R848; of which 92% is released at 2 hours and more than 96% isreleased at 6 hours. The Group 3 mice were administered NC-Niccontaining ova peptide, polymers, 75% of which were PLA-R848 and 25%were PLA-PEG-Nic, and 1.3% R848, of which 29.4% is released at 6 hoursand 67.8% is released at 24 hours. The Group 4 mice were administeredNC-Nic containing ova peptide, polymers, 75% of which were PLA-R848 and25% were PLA-PEG-Nic, and 1.4% of R848, of which 20.4% is released at 6hours and 41.5% is released at 24 hours. The Group 5 mice wereadministered NC-Nic containing ova peptide, polymers, 25% of which werePLA-PEG-R848, 50% PLA, and 25% were PLA-PEG-Nic, and 0.7% of R848; ofwhich less than 1% is released at 24 hours. Release was determined at apH of 4.5.

The results shown in FIG. 6 demonstrate that R848 adjuvant (a TLR 7/8agonist) contained in the NC augments humoral immune response againstNC-associated antigen (groups 2-5>>group 1). Furthermore, neither fast(group 2), nor slow (group 5) release of R848 was elevated an immuneresponse to the same level as NC releasing R848 at intermediate rate(group 3≈group 4>group 2≈group 5).

What is claimed is:
 1. A compound that comprises a structure as informula (IV):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; X is C, N, O, orS; R₆ and R₇ are each independently H or substituted; and R₉, R₁₀, R₁₁,and R₁₂ are each independently H, a halogen, OH, thio, NH₂, orsubstituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy, aryloxy,alkylthio, arylthio, alkylamino, or arylamino.
 2. A method for making acompound that comprises a structure as in formula (IV):

comprising: combining, in the presence of a solvent and/or heat, acompound that comprises a structure as in formula (III):

and a compound comprising a structure as in formula (V):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl; Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; X is C, N, O, orS; R₆ and R₇ are each independently H or substituted; and R₉, R₁₀, R₁₁,and R₁₂ are each independently H, a halogen, OH, thio, NH₂, orsubstituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy, aryloxy,alkylthio, arylthio, alkylamino, or arylamino.
 3. The compound of claim1, wherein R₁ is H, R₂ is isobutyl, Y is C, and R₃ and R₄ are combinedto form a benzene ring with the carbon atoms of the pyridine ring towhich they are connected.
 4. The compound of claim 1, wherein R₁ isethoxymethyl, R₂ is hydroxyisobutyl, Y=C, and R₃ and R₄ are combined toform a benzene ring with the carbon atoms of the pyridine ring to whichthey are connected.
 5. The compound of claim 1, wherein R₁ isethoxymethyl, R₂ is methanesulfonamidoisobutyl, Y=C, and R₃ and R₄ arecombined to form a benzene ring with the carbon atoms of the pyridinering to which they are connected.
 6. The compound of claim 1, wherein R₁is OH, R₂ is benzyl, Y=N, R₃ is absent, and R₄ is butoxy.
 7. Thecompound of claim 1, wherein Y is N, R₁ is OH, R₂ is benzyl, R₃ isabsent, and R₄ is butylamino.
 8. The compound of claim 1, wherein Y isN, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ is butoxy.
 9. Thecompound of claim 1, wherein Y is N, R₁ is OH, R₂ is benzyl, R₃ isabsent, and R₄ is benzylamino.
 10. The compound of claim 1, wherein Y isN, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ is pentyl.
 11. Acomposition comprising the compound of claim
 1. 12. The method of claim2, wherein R₁ is H, R₂ is isobutyl, Y is C, and R₃ and R₄ are combinedto form a benzene ring with the carbon atoms of the pyridine ring towhich they are connected.
 13. The method of claim 2, wherein R₁ isethoxymethyl, R₂ is hydroxyisobutyl, Y=C, and R₃ and R₄ are combined toform a benzene ring with the carbon atoms of the pyridine ring to whichthey are connected.
 14. The method of claim 2, wherein R₁ isethoxymethyl, R₂ is methanesulfonamidoisobutyl, Y=C, and R₃ and R₄ arecombined to form a benzene ring with the carbon atoms of the pyridinering to which they are connected.
 15. The method of claim 2, wherein R₁is OH, R₂ is benzyl, Y=N, R₃ is absent, and R₄ is butoxy.
 16. The methodof claim 2, wherein Y is N, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄is butylamino.
 17. The method of claim 2, wherein Y is N, R₁ is OH, R₂is benzyl, R₃ is absent, and R₄ is butoxy.
 18. The method of claim 2,wherein Y is N, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ isbenzylamino.
 19. The method of claim 2, wherein Y is N, R₁ is OH, R₂ isbenzyl, R₃ is absent, and R₄ is pentyl.
 20. A compound that comprises astructure as in formula (VI):

wherein R₁=H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂=H, alkyl, or substituted alkyl;Y=Nor C; R₃ is absent if Y=N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y=C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; X is C, N, O, orS; R₆ and R₇ are each independently H or substituted; R₉, R₁₀, R₁₁, andR₁₂ are each independently H, a halogen, OH, thio, NH₂, or substitutedor unsubstituted alkyl, aryl, heterocyclic, alkoxy, aryloxy, alkylthio,arylthio, alkylamino, or arylamino; and the polymer is insoluble inwater at pH=7.4 and at 25° C.
 21. The compound of claim 20, wherein thepolymer is selected from the group consisting of polyketaldiols,poly(ethylene)glycol, polycaprolactone diol, diblockpolylactide-co-poly(ethylene)glycol, diblockpolylactide/polyglycolide-co-poly(ethylene)glycol, diblockpolyglycolide-co-poly(ethylene)glycol, poly(propylene) glycol,poly(hexamethylene carbonate)diol, and poly(tetrahydrofuran).
 22. Thecompound of claim 20, wherein the polymer has a weight average molecularweight ranging from 800 Daltons to 10,000 Daltons, as determined usinggel permeation chromatography.
 23. The compound of claim 20, wherein thepolymer does not comprise polyketal or unit thereof.
 24. The compound ofclaim 20, wherein R₁ is H, R₂ is isobutyl, Y is C, and R₃ and R₄ arecombined to form a benzene ring with the carbon atoms of the pyridinering to which they are connected.
 25. The compound of claim 20, whereinR₁ is ethoxymethyl, R₂ is hydroxyisobutyl, Y=C, and R₃ and R₄ arecombined to form a benzene ring with the carbon atoms of the pyridinering to which they are connected.
 26. The compound of claim 20, whereinR₁ is ethoxymethyl, R₂ is methanesulfonamidoisobutyl, Y=C, and R₃ and R₄are combined to form a benzene ring with the carbon atoms of thepyridine ring to which they are connected.
 27. The compound of claim 20,wherein Y=N, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ is butoxy. 28.The compound of claim 20, wherein Y is N, R₁ is OH, R₂ is benzyl, R₃ isabsent, and R₄ is butylamino.
 29. The compound of claim 20, wherein Y isN, R₁ is OH, R₂ is benzyl, R₃ is absent, and R₄ is benzylamino.
 30. Thecompound of claim 20, wherein Y is N, R₁ is OH, R₂ is benzyl, R₃ isabsent, and R₄ is pentyl.
 31. A composition comprising the compound ofclaim
 20. 32. A synthetic nanocarrier that comprises the compound ofclaim
 20. 33. A composition comprising the synthetic nanocarrier ofclaim
 32. 34. A composition comprising a vaccine comprising the compoundof claim
 20. 35. A composition comprising a vaccine comprising thesynthetic nanocarrier of claim
 32. 36. A method comprising:administering the compound of claim 20 to a subject.